HIV-1 Vaccine Development: Recent Advances in the CTL Platform Spotty Business ACCEPTED

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1 JVI Accepts, published online ahead of print on 7 November 2007 J. Virol. doi: /jvi Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. HIV-1 Vaccine Development: Recent Advances in the CTL Platform Spotty Business Kimberly A. Schoenly 1 and David B. Weiner 1 * 1 The Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine * Corresponding Author 505 Stellar-Chance Laboratories 422 Curie Blvd. University of Pennsylvania Philadelphia, PA dbweiner@mail.med.upenn.edu Running title: HIV-1 Vaccine Development: Recent Advances Key words: HIV-1 Vaccine, CD8+ T cell (CTL) response, Mucosal, DNA, Viral vector, STEP trial 1

2 Introduction For human immunodeficiency virus type 1 (HIV-1) vaccine development, a central focus is to develop a vaccine that drives primarily strong and broad CD8+ cytotoxic T lymphocytes (CTLs). This is largely due to the difficulty in generating cross-neutralizing antibodies against the diverse array of HIV viral envelopes (9, 40, 78, 139). The ability of CD8+T cells to impact or control viral replication is supported by model primate studies and is reiterated by studies of immune control in infected individuals. For example, Cao et al. (28) reported that HIV-1 infected Long Term Non-Progressors (LTNP) had high levels of HIV specific CTLs. In addition, HIV-1 infected patients with high levels of virus specific CTLs exhibited lower viral loads, slower CD4+T cell declines as measured in peripheral blood, and a relatively stable clinical status (23, 81, 108, 112). Preclinical studies conducted in SIV infected monkeys showed that upon their in vivo depletion of CD8+T cells a rapid and dramatic increase in viremia was observed (70, 131). While no correlates of immunity for protection against HIV-1 have been concretely established, this data as well as others (6, 13, 137) strongly suggested that CD8+T cells are important in controlling viral infection and may be an important component of an effective HIV-1 vaccine. In this review we summarize recent advances in HIV vaccine development utilizing various platforms that target the induction of cellular immunity. In particular, we draw inferences, as appropriate, for the induction of cellular immune responses at mucosal as well as systemic sites. 2

3 CTL based vaccines- STEPped on? The discontinuation of the Merck rad5 Phase II proof-of-concept STEP study is undoubtedly a significant setback for the field of HIV vaccine development. In this study a trivalent, three vector rad5 (Gag, Pol, Nef) vaccine was tested in 2 cohorts of 1,500 individuals at high risk for HIV infection. The vaccine was scheduled for three immunizations of 1.5x10 10 viral genomes of each rad5 vector encoding Gag, Pol, or Nef administered on day 1, month 1 and month 6. There are concerns that pre-existing vector serology can mute the effectiveness of a CTL vaccine approach. Accordingly, the first cohort was made up of 1500 individuals with low levels of preexisting antibodies against Ad5, titers < 1:200, while the second cohort of 1500 had participants with anti- Ad5 titers greater than 1:200. However, the STEP study was prematurely stopped by the data and safety monitoring board because it was clear that even in the cohort with low titers of preexisting antibodies against Ad5 there was no evidence of efficacy. Table 1 summarizes the results from the cohort with titers <1:200 comparing the rates of infection and viral loads of the vaccinated versus placebo group. This was the best response group in earlier clinical studies for eliciting CTL responses in over 60% of vaccinees. As shown in Table 1, in the subset of individuals that received at least two immunizations, there was a trend toward more HIV infections in the rad5 vaccinated (19/672) as compared to the placebo (11/691) group. The viral loads taken weeks post acquisition of infection were not statistically significant between the vaccinated or placebo group, 40,000 vs. 37,000 copies per ml, respectively. Since this rad5 trivalent vaccine did not prevent infection or decrease viral replication in vaccinated individuals, the implications for CTL based vaccines remain unclear except that future studies should 3

4 test hypotheses and concepts that are not identical to the Merck approach. Furthermore, this study draws a line in the sand in that the next CTL based approach should induce higher levels of CTL responses and induce a different immune phenotype. The inclusion of different antigens or prime-boost strategies in future studies with defined immune endpoints will be important. We will consider many of these in the following sections. The Importance of Mucosal Responses The hallmark of HIV-1 infection is the rapid depletion of CD4+T cells from the peripheral blood during acute infection. More recently the ablation of CD4+ CCR5+ T cells in the gut is a dramatic consequence of both SIV (87, 99, 156) and HIV (27, 54, 102, 103) infection. The majority of HIV-1 infections occur across the mucosal barrier and the mucosal tissues are an active site for viral replication in SIV (87, 155) and HIV infection (134, 169). The induction of both antigen specific IgA antibodies and CTLs to these critical sites may provide a first-line of defense with immediate effector activity (Figure 1). Antigen specific CTLs in the mucosa could potentially eliminate infected cells within the mucosa and subsequently prevent the systemic spread of the virus. It has been shown in murine (16, 17) and macaque (18) models that mucosal, not systemic, CTLs are necessary to protect against mucosally transmitted virus. Clearly, the generation of CTLs that can target to mucosal sites may be an important facet of a successful HIV-1 vaccine. Overall there is a great need for analysis of mucosal immune responses in preclinical and clinical trials as the current available data is minimal. VIRAL VECTORS The use of viral vectors as vaccines for HIV-1 has been reviewed elsewhere (135). Viral vectors have been generally studied for gene therapy applications, but have also been 4

5 adapted for vaccine development based upon the intrinsic immunity elicited by their infection of the host. Generally, recombinant viral vectors are constructed by removing gene sequences encoding crucial viral proteins, rendering the resulting vector replication defective and using the space generated by these genes removal with vaccine antigens of interest. Recombinant viral vectors as expression systems can be engineered to transduce a wide variety of cell types, including antigen-presenting cells (APCs), and subsequently expression of HIV-1 encoded antigens in the cytoplasm of the target cell. Intracellular expression allows for the efficient priming of CD8+T cells through Major Histocompatibility Complex class I (MHCI) presentation of antigenic peptides. In addition, transduced cells serve as antigen factories, which can leak antigen for MHCII presentation. Through cross-presentation, non-transduced APCs can take up and present extracellular antigen through MHCI. APCs can bite off pieces of transduced cells (58) as well as phagocytosing apoptotic cells that contained transduced antigen (4, 15, 69, 148). It is also possible that pieces or whole antigenic protein can be transferred from transduced cells to APCs (125) and presented to CTLs through cross-presentation. In addition, viral vectors can activate innate immune responses through the pathogenassociated molecular patterns (PAMPs) of Toll-like receptors (TLRs) on APCs (reviewed in (170)). Adenovirus Adenovirus is a non-enveloped DNA virus that is typically acquired during childhood. Clinical manifestations model the common cold and include respiratory, gastrointestinal, and ocular infections. While symptoms are transient, some serotypes are capable of persisting for much longer (months-years) in mucosal tissues and continue to shed virus. 5

6 As a vaccine platform, Adenovirus has been studied extensively due to its high transduction efficiency, ease of manipulation, and ability to quickly grow to high viral titers in culture. In addition, recombinant adenoviral vectors can potentially carry large transgene inserts (up to 8kb) by deleting regions of the viral genome dispensable for viral growth (i.e. E1, E3) (21, 74). Recombinant Ad5 is rendered replication deficient by deletion of the E1 region, which is essential for adenovirus replication as well as expression of downstream viral genes. The antigenic transgene is inserted in place of the E1 region under control a promiscuous promoter like CMV and flanked by a bovine growth hormone polyadenylation terminus signal. Upon administration of rad5 vector plus antigenic insert, the recombinant viral vector binds cells expressing the coxsackievirus and adenovirus receptor (CAR) thus facilitating its internalization. As a result, antigen synthesis occurs intracellularly which leads to efficient processing and presentation of the antigen by MHCI (reviewed in (11)). A robust cellular and humoral immune response against the viral vector and transgene insert is observed in part due to its intrinsic hexon protein adjuvanting properities (104). Of all 51 known serotypes that currently exist, recombinant Ad5 has received the most attention and is currently in several clinical trials. However, the potent immune response generated against the Ad5 vector that makes it a promising HIV-1 vaccine candidate also creates one of the most difficult challenges for its development. The prevalence of preexisting anti-ad5 immunity is astoundingly high among populations that are in desperate need of an HIV-1 vaccine including many regions of Sub Saharan Africa (12, 80, 110, 158). In fact the Ad5 seroprevalence is greater than 90% in particular regions of Africa with much of the population exhibiting high neutralizing antibody titers. The Ad5 6

7 specific immunity resulting from natural Ad5 infections decreases the immunogenicity generated against the antigen insert in the recombinant Ad 5 vectors (143). In a phase I clinical trial carried out by Merck using rad5-gag, the vaccine elicited cellular immune responses in just 28% of subjects that had moderate levels of preexisting Ad5 neutralizing antibody titers (>200), whereas gag specific cellular immune responses were observed in 82% of subjects with no Ad5 immunity (reviewed in (12)). However, the trivalent vaccine at increased dosage was more effective (36). Clearly the significance of developing a vaccine to avoid preexisting antibody responses may be important and studies using chemical alterations, chimeric Ad vectors, chimpanzee Ad vectors, and rare Ad serotypes found in humans are underway. Chemical modifications to aid in Ad5 vectors immune escape include the addition of microparticles (128) or PEG (38) to reduce neutralizing antibody recognition and increase the effectiveness of multiple immunizations. Another strategy is the construction of chimeric Ad vectors, which are created by swapping regions of the Ad5 fiber or hexon proteins with corresponding regions of a different serotype. Hexon chimeric Ad vectors are capable of partially evading the antibody response against Ad5; however can be difficult to construct due to preservation of low titer neutralizing antibody epitopes (50, 150) low titer vector replication (126) and the induction of cross reactive CTLs to other proteins of the virus (167). Barouch s group constructed a rad5/ad48 chimeric vector that replaces Ad5 neutralizing antibody targets with Ad48 surface loops and has been shown to be immunogenic in rhesus macaques despite anti-ad5 immunity (122). This important modified vector is moving forward into Phase I clinical trials by Barouch s group in collaboration with IAVI (68). Novel rad5 vectors are also being constructed using 7

8 nonhuman Ads from ovine, porcine, bovine, and chimp (45, 65, 119, 120, 154, 161). In preclinical trials chimp vectors appear to be highly immunogenic with T cell responses against transgenes marginally affected by preexisting antibodies to human Ad5 (46, 117). In addition, chimp vectors elicit antibodies against the transgene in the presence of pre- existing antibodies against human Ad5, whereas antibodies against the encoded transgene-product are completely inhibited when delivered by the human Ad5 (100). Another strategy is the use of less prominent Adenovirus serotype antibodies found in humans such as Ad 6 (Reviewed in (12)), Ad 35 (158), Ad11 (67), and Ad26 (1). Specifically, its been reported that Ad35 is immunogenic and not inhibited by preexisting anti Ad5 antibodies in mice (12); however Ad35 elicits less potent anti-transgene responses than recombinant Ad5 vectors (11, 136). Ad35 is in clinical development by the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), and GenVec. Based upon the safety data of Ad35 in 15 volunteers, a second part of the trial will pursue the delivery of Ad35 in combination with Ad5 (68). Ad26 is another adenovirus serotype less prevalent in humans that will be moving into clinical trials by Barouch s group and IAVI (68). In rhesus macaques, rad26 elicited strong cellular and humoral immune responses similar to those observed with rad5 (1). In addition, an rad26 prime/rad5 boost elicited cellular responses 10 times that observed with a homologous rad5 immunization, and controls viremia following a SIVmac251 challenge (84). Recombinant Adenovirus vectors are also attractive vaccines for HIV-1 because they can be administered at mucosal sites- intranasal and orally. Furthermore, they have an intrinsic tropism for mucosal tissues where they can persist for some time. In mice, Adenoviral vectors have been shown to elicit cellular immune responses in the 8

9 mucosa (92, 147); however there is limited mucosal data from nonhuman primates using these vectors and it will be important to further characterize these responses. One STEP forward- Lessons learned for nonhuman primate studies Studies comparing cellular data between recombinant adenoviruses, poxviruses, alphaviruses, and plasmid DNA vaccines all expressing SIV Gag supported that rad5 elicited the highest levels of antigen specific CTLs at the time the trials were decided (136, 137). The results of rad5 in animal models highlight considerations that should be considered in the evaluation of candidate vaccines. The rad5 vaccine never protected against infection: regardless of the monkey haplotype or challenge virus studied all monkeys became infected following challenge, leading to the unrealistic expectation that vaccination with the rad5 trivalent vaccine would protect against infection in humans. There was a clear hierarchy of control of viral replication by rad5 vaccination in primate challenge models. In hindsight, the easiest challenge model, A01+ macaques challenged with SHIV 89.6p, produced a clear 3 log reduction in viral loads compared to control (137). The more virulent SIVmac239 challenge resulted in a sustained1.5 log decrease in viral loads of rad5 immunized A01+ monkeys (101). It is clear that Mamu A01+ rhesus macaques are useful models for observing antigen specific responses using tetramer staining, but it is likely that they are not accurate models for predicting vaccine efficacy for humans. In preclinical studies with the rad5 vaccine testing outbread macaques challenged with SHIV89.6, a more variable 2-3 log reduction was observed (89). However, when outbred macaques were challenged with SIVmac239 post rad5 immunization, no significant reduction in viral load was seen in vaccinated monkeys compared to control (29). This hierarchy establishes that the use of outbred macaques 9

10 and SIV challenge should be considered the most stringent vaccine model and be a focus for future evaluation of HIV vaccine efficacy studies. However, based on a lack of evidence of protection competency in this model it is clear that all primate studies are still at risk studies without known correlates or clear immune targets. One STEP back- Implications CTL based vaccines Merck, the HIV Vaccines Trial Network (HVTN), and Department of AIDS (DAIDS)/NIH should be commended for their valiant effort, creativity, organization, and management of the important large-scale efficacy trial. The bar for the STEP study set high expectations as rad5 was the most promising CTL platform based upon clinical and preclinical data in rhesus macaques. In earlier clinical trials, significant response rates among low Ad5 seropositive individuals vaccinated three times with the trivalent Ad5 were observed to all three HIV antigens at week 30: 73% to Gag, 59% to Pol, and 68% to Nef. The reported geomean of responders as measured by IFN-γ ELISpots were 205, 342, and 177 spot forming units (SFU) to Gag, Pol, and Nef respectively (37). Response rates were lower in high titer Ad5 seropositive individuals at the same timepoint with 69% to Gag (406 SFU), 38% to Pol (834 SFU), and 54% to Nef (428 SFU) (37). The majority of responders only responded to one eptitope in gag, pol or nef peptide pools, with approximately 30% of responders recognizing 3 or more epitopes (37). Although the trial primarily consisted of men who have sex with men (MSM), it is interesting that a very small number of women were infected in this trial. This should be closely watched as it may indicate that vaccine elicited immune responses may differ between genders. Such an observation has implications for MSM versus heterosexual transmission. With this in mind, the analysis of the responder data in from STEP trial will be critical in 10

11 determining a relationship in vaccine responders, the level of the response, and the resulting incidence of infection. In addition, we anxiously await the data analysis from the high titer vector seropositive cohort to see if these results reflect the same trend of infection states observed in the low Ad5 seropositive cohort. The increased trend toward infection is not likely a general trend for all vaccines that elicit T cells. The Vaxgen protein trial, which mobilized B and T helper cells did not have a similar trend. The ALVAC trials, which mobilized weak CD8+T cells similarly did not report this trend. This trend it should be noted is not significant at this time and therefore it is importance to vaccines under development remains speculative. Unanswered questions include: Are the response levels (on average about 350 SFU) observed in responders high enough? Are the vaccine targets the most relevant? Would polyfunctional immune responses or some other T cell phenotype change the vaccine efficacy? Do the responses need to be directed against a broader number of epitopes to be effective? What animal models are most important as predictors of human vaccine efficacy? What do the mucosal responses look like in Ad5 vaccinated people, and would such responses change the impact of the vaccine? Adeno-associated virus Adeno-associated virus (AAV) is a helper dependent parvovirus that has been well characterized as a gene therapy vector (reviewed in (144)). The generation of the recombinant vector entails removing all viral genes except the sequences that encode the GC-rich inverted terminal repeats (ITRs). The entire genome is a mere 4.7kb, which limits the size of the antigenic insert (between the ITRs) to roughly 3.8kb, excluding the promoter and polya tail. The removal of viral genes reduces the intrinsic 11

12 immunogenicity of AAV, however, raav remains an attractive vaccine candidate due to commercial scale production ability (34), outstanding safety profile in the clinic for nearly a decade (reviewed in (72)), ease of intramuscular administration and high transduction efficiency of myofibers (reviewed in (72)). Johnson, et al. (72) demonstrated the efficacy of raav for delivery of SIV antigens in an intravenous challenge (SIVsm/E660) model. A portion of their study included two injections of three raav vectors encoding SIV genes at doses measured by DNAse resistant particles (DRPs), which are equivalent to encapsulated vector genomes. The three raav vectors contained fragments of SIV Gag, protease, and reverse transcriptase (1) SIV rev, env (2) and reverse transcriptase, integrase (3) at10 13, 5 X 10 12, and DRPs, respectively. The results show a robust cellular (tetramer specific CTLs) and humoral (serum antibody) immune response after a single injection. In fact, after a virulent intravenous challenge, immunized monkeys exhibited significant control of SIV replication with viral loads almost 2 logs lower than the control group. The protection from challenge may be attributed to CTLs as there were no detectable neutralizing antibodies against the SIV challenge stock observed in the serum of vaccinated animals. Interestingly, there was no increase in the observed immune response after the second immunization, suggesting the generation of anti-vector antibodies. In fact, the preexisting or vaccine vector-induced immunity to capsid is still a major concern for this platform. For example, recombinant AAV2 is the most studied serotype to date and it has been shown that 27% of the population has neutralizing antibodies directed against AAV2 (162). Similar to strategies being employed with recombinant adenoviral vectors, less prevalent raav serotypes found naturally in humans are being examined including AAV1 and AAV5 to circumvent 12

13 this issue. Alternating raav capsid serotypes from one immunization to the next is being explored. A Phase II clinical trial organized by Targeted Genetics and IAVI utilizing raav encoding HIV gag, pol, reverse transcriptase (tgaac09) is underway in Zambia, and has been reported safe and well tolerated thus far. While initial clinical studies using raav appear to be overwhelmingly safe, recently a patient participating in a Phase I/II trial gene therapy trial treating rheumatoid arthritis died after receiving a raav vector encoding the soluble TNF-α receptor, tgaac94. The patient was also taking a TNF-α blocker, Humira, which is immune-suppressive. While no definitive reports have determined the cause of the patient s death, it appears likely that a fungal infection, Histoplasma capsulatum, could be the cause (75). It is not clear the role of the AAV treatment in this patient s outcome. Importantly, this study as well as the Ad5 STEP study highlight the potential pitfalls of performing trials in immunosuppressed individuals whose responses are clearly different from immunocompetent people. The immunogenicity of raav vectors clearly needs improving, and the use of molecular adjuvants and prime boost strategies will undoubtedly enhance the efficacy to this approach and will be discussed in future sections. The published data on raav generation of mucosal immune responses is limited although it is an interesting vector due to its stability at a wide range of ph and high temperatures. Alphaviruses & Rhabdoviruses Alphaviruses are positive stranded RNA viruses with a broad host range that cause a variety of mosquito-transmitted diseases including Eastern Equine encephalitis (EEE), Western Equine encephalitis (WEE), and Venezuelan equine encephalitis (VEE) in the United States. While the majority of alphaviral infections are asymptomatic in humans, 13

14 flu-like symptoms can occur including fever, head- and body-aches. The virus is also capable of invading the central nervous system leading to encephalitis and perhaps even death. Modifications to the virus for vaccine vector generation has been well studied (reviewed in (118, 130). To generate vaccine replicon particles, the nonstructural protein genes and natural subgenomic promoter (26S) are retained while the structural protein genes are replaced with the antigenic gene of interest. Current alphavirus replicon particle vaccines have been derived from the Sindbis virus (SIN), Semliki Forest virus (91), and VEE. This creates a single cycle replication vector that is translated upon entry into the cytoplasm of the target cell, allowing for intracellular processing and subsequent efficient presentation by MHCI. The cytoplasmic amplification of these vectors includes a double-stranded RNA intermediate that activates both the innate and adaptive immune system, strengthening this platform as a vaccine candidate delivery system (85). Indeed the replication of alphavirus replicons results in extremely high levels of antigen expression (157, 166, 171); however, this replication is generally toxic to the infected cell thus preventing long-term antigen expression (48). The anti-vector immune response against Alphavirus replicons has so far been minimal allowing for re-administration, a major benefit for a vaccine (118). Similarly, the seroprevalence is low in humans and the replicons can be engineered to be lymph node trophic, enhancing antigen presentation through efficient targeting to and expression in the immune inductive site. A possible drawback to the alphavirus platform includes the requirement of the rep protein for highlevel antigen expression. This antigen may be prone to immune surveillance by CTLs. However, this platform has many positive features. For example, previous studies report the primary cellular target of replicon particles has been shown to be those of the CD14+ 14

15 monocyte lineage in rhesus macaque (55) and dendritic cells in human peripheral blood mononuclear cells (PBMCs) (51). In nonhuman primates vaccination with 2x10 8 PFUs of chimeric SINenv and VEErep alphavirus replicon particles expressing SIVp55Gag and/or HIV V2gp140 alone resulted in high titers of anti-hiv neutralizing antibodies, while moderate anti-env IFN-γ ELISpot responses were also observed (163). In a separate study, 1x10 7 infectious units of each of three VEE replicon particles (VRPs) expressing the SIVsmH-4 matrix-capsid region of Gag, gp160 or unanchored gp140 were administered three times to rhesus macaques followed by a mucosal challenge with SIVsmE660 (73). Macaques immunized with SIV-VRP produced neutralizing antibodies and had reduced peak viral load (1 log) post-challenge. Correlating with decreased viral loads was an increase in peripheral CD4+T cell counts of immunized macaques observed at viral set point. While not measured in this study, it will be interesting to analyze immune responses at mucosal sites, namely the preservation of CD4+ T cells. Overall, however, this platform has progressed very slowly. To date there is a limited amount of clinical data and studies to prove the efficiency of this platform are clearly important. Vesicular Stomatitis Virus A member of the Rhabdoviridae family includes Vesicular stomatitis virus (VSV), a negative strand RNA virus that primarily infects livestock. In most of the world, human infection is rare and asymptomatic, but can cause flu-like symptoms. VSV is an attractive platform for the clinic as it can be delivered at mucosal sites, carry multiple transgenes, is easily manufactured, and has low seroprevalence in humans (reviewed in (88, 121)). One potential drawback to VSV vaccine vectors is the development of antibodies against VSV surface glycoproteins in immunized hosts after one 15

16 immunization. Therefore, effective boosting may require swapping in a glycoprotein from a different VSV serotype. In non-human primates, immunization with rvsv vectors encoding HIV env and SIV gag protected against disease post SHIV89.6p challenge (41, 43). Furthermore, the direct comparison of intramuscular versus intranasal administration of equal doses of HIV env and SIVgag (5x10 6 PFUs each) in rhesus macaques demonstrated that intranasal vaccination elicited higher cellular immune responses as measured by IFN-γ ELISpot, Cr- release assays, and tetramer analysis (43). Interestingly, there was no difference in the humoral immune responses elicited by either vaccination strategy at systemic or mucosal sites (43). The intranasal delivery of VSV vectors does not seem to cause neurovirulence in non-human primates (71). It will be interesting to see if the mucosal administration of this promising platform can elicit cellular immune responses at mucosal sites. Poxviral vectors Poxviruses are large DNA viruses that evolved based on experience with the smallpox vaccine (reviewed in (93)). As vaccine vectors they are able to carry multiple and large genes (~25kb) (97) that are stably expressed. These vectors are also capable of inducing immune responses similar to those observed with the pathogen. Several studies showed that vaccina vectors can be pathogenic in immunocompromised people thereby refocusing efforts on developing a more attenuated, modified Vaccinia virus Ankara (MVA) as a vector system (105). MVA has been shown to be safe in mice, monkeys, and humans as it cannot complete an entire life cycle in humans or primates (41) while still being capable of inducing a strong cellular and humoral immune response in mice and primates. Numerous poxvirus-based vaccines have been tested in rhesus macaques after 16

17 challenge with SIV, SHIV, and HIV-2 isolates ((2, 7, 19, 47, 57, 59-62, 114) and have shown various degrees of cell-mediated immune responses and protection against challenge. A study conducted in rhesus macaques delivered SHIV DNA plasmid producing noninfectious viral particles intranasally with a rmva SIVgag-pol and HIVenv boost. Co-immunization with a plasmid IL-2/Ig elicited both mucosal and systemic humoral and cellular anti-shiv immune responses (22). Intranasal immunization with other platforms has been previously reported to be the only route of mucosal vaccination that results in disseminated humoral and cellular immune responses (82, 83) and results in increased vaginal responses over rectal immunization (20, 83). Indeed, nasal immunization also induced greater systemic antibody responses and thus to generate both systemic and mucosal immunity, intranasal may precede other mucosal routes (83). Mucosal SHIV specific responses were detected in the IL-2/Ig group as measured by IgA in rectal secretions as well as tetramer positive T cells in rectal biopsies. Upon rectal challenge with SHIV89.6p all animals became infected, but the IL-2/Ig co-immunized group was protected from CD4+T cell loss and disease progression to AIDS. Interestingly, a different study showed the induction of tetramer-positive CD8+ T cell responses at mucosal sites whether live attenuated poxvirus NYVAC encoding SIVgag, pol, env was administered by intranasal, intramuscular, or intrarectal routes (140). A recombinant canarypox vector (ALVAC 1452) has been tested in multiple clinical trials with different doses, formulations, and prime/boost regimens by the HVTN and others. The results of a Phase II study of ALVAC and VaxGen s gp120 protein (HVTN203) demonstrated less than 36% vaccinees responded in an IFN-γ ELISpot 17

18 assay, and therefore the planned Phase III trial (HVTN501) was cancelled. Results from another Phase II trial testing ALVAC with or without a rgp120 boost outside of the U.S. (HVTN 026) did not show a difference in cellular immunity as measured by IFN-g ELISpot, lymphocyte proliferation assays, or Cr release assays between vaccinated and placebo groups (35). Increasing the dose of ALVAC vector (HVTN 039) or the addition of lipopeptides (HVTN042) did not enhance the number of responders, 5/52 or 3/77 respectively (37). A phase III clinical trial in Thailand involving the same ALVAC vector expressing subtype E env and subtype B env (gp41), gag, protease plus gp160 or Chrion gp120b/e boosting is currently underway (76). In this study, CTL response rates are observed in less than 30% of recipients and antibodies are observed, although broadly neutralizing antibodies are not. This may have been expected as the preceding Phase I/II study results showed no statistical difference between cellular responses in the ALVAC prime plus gp120 or gp160 protein boost vaccinated and placebo groups (149). Improving the immunogenicity of these experienced vectors is a major focus of the poxviral field. Herpesvirus Unlike the viral vectors described above, herpes simplex virus type 1 (HSV-1) is able to persist for essentially the lifetime of the infected host. Active HSV-1 infection occurs at mucosal surfaces including the mouth and genitals, and the virus remains latent in the nervous system where it can be periodically reactivated. In addition, both cellular and humoral immune responses are generated in response to HSV-1 infection. These features of HSV-1 highlight its potential utility as a vaccine vector in regard to long-term antigenic expression, immunogenicity, and tropism for mucosal surfaces. Both 18

19 replication- competent and replication- defective recombinant HSV-1 vectors have been studied as vaccines for HIV-1 in nonhuman primate models. In a study by Murphy, et al. (107) the replication capability of HSV-1 did not elicit statistically different results as measured by antibody levels, CTL activity or protection against challenge. In fact, 2 rhesus macaques were protected against a SIVmac239 rectal challenge- one immunized with a replication- competent and the other a replication- deficient HSV-1 encoding SIV Env and Nef. However, the replication- competent group had peak viral loads 1.2 logs lower than infected control animals. Clearly, the protection against mucosal challenge and tropism of HSV-1 for mucosal surfaces warrants further examination of immune responses elicited by the mucosal delivery of this platform. BACTERIAL VECTORS The use of recombinant bacteria as vectors for gene delivery has been extensively reviewed elsewhere (94). Here we will touch on two particular platforms that have been used for HIV-1 vaccines in nonhuman primate models- Listeria monocytogenes and Salmonella. Listeria monocytogenes The use of a recombinant Listeria monocytogenes as a vaccine vector has many promising features including a natural tropism for mucosal tissues, an ability to infect APCs, high CD8+T cell induction, low prevalence of pre-exposure in humans, and it s ease of manipulation and production in culture (Reviewed in (90)). Two independent studies performed in rhesus macaques illustrated the induction of mucosal cellular immune responses with a DNA prime, Listeria boost, both encoding SIV Gag and Env (26, 109). In the study performed by Boyer, et al. (26) the macaques that received the 19

20 DNA prime-listeria boost had better protection against an intrarectal challenge of SIV239, as measured by a control of viral loads for a longer period of time. Neeson, et al. (109) further examined the DNA prime-listeria boost induction of a mucosal SIV gag specific cellular immune responses by demonstrating Gag specific CD8+T cells in the peripheral blood co-express the gut-homing marker, B7, in the peripheral blood, and home to gut mucosal tissues. Salmonella Like Listeria monocytogenes, the natural route of infection is oral, and therefore use of the recombinant bacteria to induce mucosal immunity is promising. Recombinant Salmonella vectors have been engineered to utilize the bacterial type III secretion system in order to more efficiently present antigenic peptides via MHCI and subsequently prime CD8+ CTLs (127). Priming the immune response with an intragastric administration of the modified Salmonella vector expressing HIV-1 gag in combination with a MVA gag boost, mucosal immune responses were elicited in rhesus macaques (44). This combination in particular elicited SIV gag specific CD8+T cells that expressed α4β7 and homed to the colonic mucosa. While such responses were unable to confer protection against an intrarectal challenge with SIV239, the elicitation of mucosal cellular responses (albeit minimal) is noteworthy. The authors speculate that with addition of more antigens to the Salmonella vector, a more robust and diverse mucosal cellular response could be achieved. 20

21 NUCLEIC ACIDS While RNA has been important in the development of immune therapy for cancer, there is limited work in the HIV-1 vaccine area in particular. Accordingly we focus here on the DNA approach. As a vaccine platform, DNA has been studied for over 15 years. Studies in mice immunized with plasmid antigens yielded promising levels of immunity that unfortunately have not been well translated in initial clinical studies. Currently, there is a significant amount of work in the field devoted to improving this platform as it is appealing on many levels including large-scale vaccine production, safety, repeat administration (no preexisting or vaccine induced vector serology), and storage as a cold chain is not required. DNA vaccines for HIV-1 have been reviewed elsewhere (66). Here we will focus primarily on optimization of future DNA vaccines. Genetic Level The optimization of plasmids has yielded a substantial increase in the immunogenicity of plasmid-encoded antigens via enhanced levels of gene expression on a per cell basis. For example, codon optimization improves the expression by adapting the codon usage to the bias of the target species, thereby enhancing transcription through more abundant trna pools (8, 39, 159, 172). Additional modifications may also be made to the encoded gene sequence to increase the stability of the mrna including eliminating mrna instability and mrna inhibitory elements (133). The inclusion of a Constitutive Transport Element (CTE) can enhance RNA stability and export from the nucleus. The addition of IgE leader sequences also facilitates expression by improving mrna loading onto the ribosome (159, 164). By removing these negative sequences, avoiding splice sites, 21

22 removing double stranded pairing/secondary structure formation and including the above sequences, mrna structures are created that are more stable and more efficiently transported to the ribosome. As a result the mrna is translated more effectively and gene expression is enhanced on a per cell basis, which can improve immunogenicity. Molecular Adjuvants As a vaccine strategy, the immunogenicity of DNA has been suboptimal. In addition to genetic modifications enhancing the expression of the antigenic plasmid, there is a plethora of work on improving the potency of DNA vaccines. One promising technique involves the incorporation of plasmids encoding immune modulatory genes. Molecular adjuvants reported on for the induction of mucosal immunity include chemokines CCL19, CCL21, MIP-1α and cytokines GM-CSF, Il-2, IL-12, and IL-18 (152). Two recent nonhuman primate studies conducted by Wyeth and colleagues included the plasmid-encoded cytokines IL-12 (129) and/or IL-15 (33) co-delivery with an SIV gag DNA construct. Plasmid IL-12 was the best adjuvant, substantially enhancing both cellular (ELISpot) and humoral (ELISA) immunity against HIV-1 gag. Co-immunization with both IL-12 and IL-15 enhanced humoral, but not cellular responses and this version of the IL-15 plasmid did not statistically enhance responses seen with antigen alone. Upon challenge with the pathogenic SHIV89.6p virus, macaques co-immunized with IL- 12 had lower viral loads and a trend toward the preservation of peripheral blood CD4+ T cells although the groups were too small for statistical significance. Similarly, coimmunization with plasmid IL-12 augmented cellular immune responses as measured by IFN-γ and granzyme B in cynomologus macaques (24). In a separate study, cynomologus macaques co-immunized with plasmid encoded SIVgag and a highly 22

23 optimized macaque IL-15 had significantly lower viral loads compared to the antigen only group following a SHIV89.6p viral challenge, even though co-immunization with IL-15 did not significantly enhance the IFN-γ response over antigen alone (25). This study as well as others highlights the necessity to supplement the ELISpot assay with multi-parameter immune response analysis, as well as other assays of T cell function. Another adjuvant IL-2 has shown encouraging potential. Co-immunization of rhesus macaques with a plasmid gag and env with an IL-2/Ig fusion construct resulted in a potent CTL response and subsequently reduced viral loads, maintained stable CD4+T cell counts, and prevented disease progression to AIDS (13). Overall, these primate data are exciting. The clinical studies of such approaches are anxiously being followed. In addition to cytokines being co-delivered with antigenic plasmid, other immunomodulatory genes such as co-stimulatory molecules are being tested in macaques including CD40L and GITR (141, 142) and GM-CSF (14), B7.1, LFA-3, and ICAM-1 (165) in mice. Whether there are mucosal benefits to DNA vaccination remains to be seen. Delivery Improvements Strategies to improve the dosing, delivery, longevity, and transfection efficiency of DNA vaccines are receiving a great deal of attention. One method of facilitating delivery of plasmid DNA is through formulations including incorporation of liposomes or polymers to extend plasmid longevity following injection and thereby enhancing uptake over time and subsequent expression (5, 32, 53, 111). Improved delivery methods include the gene gun (49, 145, 153), BioJet (3, 56, 153), laser (168), ultrasound (146), and electroportation (63, 151, 160) approaches to enhance physical delivery. The gene gun is advantageous in 23

24 that it requires very low amounts of DNA to be effective. This method of plasmid delivery has an apparent ability to drive antibody responses in both nonhuman primates and humans, although improving CTLs is an important goal of this technology. Another strategy, the BioJet is a needle-free jet injection device that may reduce variability of responses in primates and humans (3, 56, 153). The use of laser, ultrasound, and electroporation are all based on the permeabilization of cell membranes at the injection site through light, sound, and electric current respectively. Various types of in vivo electroporation have also been recently employed to increase transfection efficiency of plasmid DNA. This technique involves applying a specific electric current to the target either the muscle or skin based upon the type of immunization delivered- intramuscular or intradermal. Enhanced delivery is observed and is likely due to the formation of transient pores within the cell membrane, allowing for more cellular uptake of plasmid DNA. Concurrently, an inflammatory response is induced and likely enhances the observed immune response as well (10). In a comparative study of intramuscular delivered plasmid DNA (IM) versus electroporation (IM/EP), electroporated mice were given 2 to 10 fold less DNA compared to the IM group. As shown in Figure 2, electroporated mice immunized with 25ug of DNA had levels of IFN-γ secretion 2 fold higher than the IM group immunized with 50ug of DNA without electroporation (approximately 550 versus 250 SFC, respectively). Furthermore, electroporated mice immunized with only 5ug of DNA had levels of IFN-γ secretion similar to mice immunized IM with 50ug and elicited significantly higher anti-gag antibody levels compared to IM without electroporation (Figure 2). In rhesus macaques immunized with plasmid DNA versus electroporation with one-fifth of the DNA, the 24

25 electroporated group had enhanced cellular immune responses by fold with antibody titers 2.5 logs greater than DNA alone (96). The results of this study help to put plasmid DNA vaccines for HIV-1 on the potency map. Benefits as seen in the primate model include enhanced plasmid delivery resulting in increased gene expression as well as enhanced immunogenicity (64, 113). The combination of electroporation with novel formulations is exciting to consider. Our laboratory has shown a significant enhancement of T cell proliferation in rhesus macaques that were given an intramuscular DNA immunization with electroporation versus an intramuscular immunization alone (L. Hirao, L. Wu, A.S. Khan, D.A. Hokey, J. Yian, A. Dai, M.R. Betts, R. Draghia-Akli, and D.B. Weiner, submitted for publication). However, there are obvious concerns with this technology versus the standard needle & syringe delivery method including: increased pain, more complex technology to establish, requirement for a machine and training for the clinical staff who administer the vaccine. The overall goal will be to decrease the required dose and voltage and subsequently increase the tolerability for an effective vaccine. The inclusion of cytokines appears to further lower the dose while simultaneously enhancing the potency of the vaccine, and may allow for a decrease in the number of required injections to achieve useful clinical potency. Prime-boost protocols Heterologous prime-boost protocols are particularly beneficial for recombinant viral vectors as anti-vector immune responses can prevent the readministration of the homologous vector. DNA is a conceptually weak delivery system that is specific for delivery of the antigen. Most studies have shown that DNA can prime efficiently for protein or recombinant viral vector boost. 25

26 DNA/Vector prime-protein boost The administration of a DNA prime and subsequent protein boost effectively induces both cellular and humoral immunity. In rhesus macaques, a polyvalent DNA encoding Env from multiple subtypes and a gag gene prime plus homologous gp120 proteins boost elicited modest cellular (around 500 SFC) and robust humoral responses (~10 6 ) (116). Upon rectal challenge with SHIV-Ba-L, the majority of immunized macaques were protected from infection while the rest had lower viral loads than the unvaccinated group. A similar human ALVAC-HIV-1 recombinant vaccine expressing gag, pol and gp120 or gag, pol and gp160 prime plus a homologous env protein boost resulted in decreased viral load levels in the blood and in mucosal sites while simultaneously protecting macaques from peripheral CD4+T cell loss after challenge with SHIV KU2 (115). The promising nonhuman primate data is being studied in a Phase I clinical trial using five different gp120 isolates and one gag DNA prime plus a boost with heterologous gp120 protein by Lu, et. al (95). Interestingly, anti-gp120 antibody titers were observed comparable to those seen in patients with chronic HIV-1 infection. In addition, anti-env cellular responses were induced by this combination. DNA prime-rad boost Recombinant Ad5 is also being used as a boost for a unique DNA prime in large-scale clinical trials being conducted by the NIH and VRC. In a Phase I trial, DNA and Ad5 were administered separately at increasing doses to 86 recipients. The vast majority of recipients made an anti-hiv-1 T cell response- the first published reports of high responses (30, 52). Four DNA constructs Clade B gag-pol-nef fusion protein, and Clades A, B, C Env glycoproteins (31, 79) elicited antibody, and long lasting env-specific 26

27 CD4+ and CD8+ T cells. Comparatively, a higher percent of responders with greater magnitude immune responses was observed in recipients of rad5 (Clade B Gag-Pol fusion protein, Clades A, B, C env glycoproteins). Although impressive levels of cellular and humoral immunity were observed in the majority of subjects, preexisting immunity to Ad5 lowered T cell responses approximately 3 fold as compared to seronegative subjects. Based on the safety and encouraging results from this study, a Phase II trial has been initiated (124). Recent studies have shown a dramatic increase in HIV-1 specific immune responses with the combination of these two platforms. The DNA prime with rad5 boost resulted in greater than 1000 fold increase in antibody and greater than 5 fold T cell responses against env (124). Data from macaques immunized with this strategy and subsequently challenged with SIV shows a long-term preservation of total central memory CD4+T cells number and function in the peripheral blood as a result of a vaccine generated protection against high levels of virus replication (86, 98). Efficacy trials of this approach are being planned through HVTN/VRC collaboration. DNA prime-raav boost In the raav nonhuman primate study previously mentioned by Johnson, et. al (72), a DNA prime/raav boost protocol elicited the most potent responses compared to other regimens using raav vectors alone. The study utilized either a DNA or raav prime with a mixture of three plasmids encoding the same SIV subgenomic fragments of gag, protease, and reverse transcriptase (1), Reverse transciptase and integrase (2) and rev and env (3). Both groups received the same raav rev-env boost. The monkeys with the lowest viral loads and therefore best control of SIV replication post challenge were in the DNA prime and raav boost group despite the weakly immunogenic DNA prime. 27

28 DNA prime-vsv boost While preexisting immunity to VSV is not an obstacle in humans, the ability to boost with the same rvsv strain is limiting due to anti-vector immune responses. Therefore, the use of VSV in a heterologous prime-boost regimen with plasmid DNA is an attractive strategy for eliciting more potent immune responses. In A01 negative rhesus macaques, intramuscular immunization of 5mg each DNA plasmid encoding SIVgag and cytokine adjuvant IL-12, followed by intranasal delivery of 5x10 6 PFUs of each rvsv encoding HIVenv (gp120) and SIVgag boost elicited both cellular and humoral immune responses greater than either platform alone (42). In particular, following a high dose (300 monkey infectious doses) intravenous SHIV89.6p challenge, this prime-boost regimen resulted in a 2.4 log reduction in viral loads compared to the DNA prime (1.5 log) or rvsv boost (1.1 log) given alone (42). In addition, there was enhanced preservation of CD4+ T cells in the peripheral blood of these animals in addition to no observed clinical disease post challenge (42). DNA prime-mva boost One of the most studied prime-boost regimens is DNA prime and poxvirus boost (123, 132). Interestingly, the T cell responses generated with this heterologous strategy give immune responses 10 times higher than either platform given separately (106). In fact, a promising clinical trial underway by Robinson, et al. and the HVTN utilizes a multivalent DNA (Gag, Pol, Env, Tat, Rev, Vpu) prime with a MVA boost. Initial results in humans show exceptional safety and tolerability, but undetectable HIV-1 specific cellular (responders= 0/28 by IFN-γ ELISpot and 0/24 by Cr-release (37)) or humoral responses after a DNA prime (106). However, based upon data in rhesus macaques, animals with 28

29 minimal responses detected after DNA prime were able to generate a potent immune response post MVA boost (138) suggesting the same may be true in humans. Preliminary reports suggest the poxviral component is increasing the levels of immunity observed in these studies. In addition, data from another rhesus macaque study using a gag, Pol, Env DNA prime plus MVA boost resulted in the control of viremia after a mucosal 89.6p SHIV challenge (6). DNA prime-hsv-1 boost A recent study in rhesus macaques compared immune responses elicited by immunization of recombinant DNA and HSV-1 encoding SIV genes in a heterologous DNA prime- HSV-1 boost strategy to homologous HSV-1 immunizations (77). Despite robust cellular and modest humoral immune responses observed in immunized macaques, all became infected upon an intravenous challenge with SIV239. Interestingly, the DNA prime- HSV-1 boost immunized macaques had levels of T cell responses greater than macaques immunized with HSV-1 alone. The observed levels of IFN-γ secretion in the prime-boost group were greater than or equal to those observed in unvaccinated macaques infected with wild-type SIV. In fact, while the DNA prime- HSV-1 boost also had a higher percent of tetramer-postive IFN-γ secreting cells, the group that received homologous HSV-1 immunizations had more α4β7 positive cells, a marker of T cells homing to the mucosa. We look forward to future studies addressing the ability of HSV-1 to induce mucosal immune responses. Conclusions It is a tumultuous time for HIV vaccine development. There are few promising leads in the vaccine toolbox for antibody based approaches. The STEP study has thrown cold 29

30 water on the cadre of new CTL based platforms. These approaches provide the broadest array of T cell driving vectors in the history of vaccinology. In the near future adjuvanted DNA, additional prime-boost studies, designer Ad vectors, and DNA electroporation strategies will be expanded in the clinic. The world s first glimpse of the lack of efficacy of a pure T cell based approach brings forth new research goals for the HIV vaccine field. 1) More stringent primate models should be utilized in determining vaccine efficacy, namely the exclusion of natural controlling animals in challenge decision studies. 2) We should demand more CTL s, greater than an average of ELISpots from next generation approaches. 3) The induction of T helper responses are desirable and likely important. 4) The importance of polyfunctional immune responses in addition to proliferative capacity of vaccine elicited T cell responses should be studied as it relates to vaccine outcome in macaques. 5) Novel and clean new hypotheses for vectors entering clinical trials should be encouraged and immune evaluation designed on this basis. 6) Most importantly Congress should support NIH by increasing HIV vaccine basic research funding and early pilot clinical testing programs. New ideas and concepts are critical and cannot be advanced effectively based on current funding levels for R01 s and P01 s. The Merck STEP study should be a catalyst for propelling the next generation of vaccines forward by raising the bar for what we expect from them. Such an occurrence will firmly add the CTL platform with all its potential promise to the armitarium of vaccinologists,k and with some hard work and luck welcome in the heyday of the age of designer vaccine platforms. 30

31 REFERENCES 1. Abbink, P., A. A. Lemckert, B. A. Ewald, D. M. Lynch, M. Denholtz, S. Smits, L. Holterman, I. Damen, R. Vogels, A. R. Thorner, K. L. O'Brien, A. Carville, K. G. Mansfield, J. Goudsmit, M. J. Havenga, and D. H. Barouch Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. J Virol 81: Abimiku, A. G., G. Franchini, J. Tartaglia, K. Aldrich, M. Myagkikh, P. D. Markham, P. Chong, M. Klein, M. P. Kieny, E. Paoletti, and et al HIV- 1 recombinant poxvirus vaccine induces cross-protection against HIV-2 challenge in rhesus macaques. Nat Med 1: Aguiar, J. C., R. C. Hedstrom, W. O. Rogers, Y. Charoenvit, J. B. Sacci, Jr., D. E. Lanar, V. F. Majam, R. R. Stout, and S. L. Hoffman Enhancement of the immune response in rabbits to a malaria DNA vaccine by immunization with a needle-free jet device. Vaccine 20: Albert, M. L., B. Sauter, and N. Bhardwaj Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392: Alpar, H. O., S. Somavarapu, K. N. Atuah, and V. W. Bramwell Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery. Adv Drug Deliv Rev 57: Amara, R. R., F. Villinger, J. D. Altman, S. L. Lydy, S. P. O'Neil, S. I. Staprans, D. C. Montefiori, Y. Xu, J. G. Herndon, L. S. Wyatt, M. A. Candido, N. L. Kozyr, P. L. Earl, J. M. Smith, H. L. Ma, B. D. Grimm, M. L. Hulsey, J. Miller, H. M. McClure, J. M. McNicholl, B. Moss, and H. L. Robinson Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292: Andersson, S., B. Makitalo, R. Thorstensson, G. Franchini, J. Tartaglia, K. Limbach, E. Paoletti, P. Putkonen, and G. Biberfeld Immunogenicity and protective efficacy of a human immunodeficiency virus type 2 recombinant canarypox (ALVAC) vaccine candidate in cynomolgus monkeys. J Infect Dis 174: Andre, S., B. Seed, J. Eberle, W. Schraut, A. Bultmann, and J. Haas Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. J Virol 72: Apetrei, C., P. A. Marx, and S. M. Smith The evolution of HIV and its consequences. Infect Dis Clin North Am 18: Babiuk, S., M. E. Baca-Estrada, M. Foldvari, M. Storms, D. Rabussay, G. Widera, and L. A. Babiuk Electroporation improves the efficacy of DNA vaccines in large animals. Vaccine 20: Barouch, D. H., and G. J. Nabel Adenovirus vector-based vaccines for human immunodeficiency virus type 1. Hum Gene Ther 16: Barouch, D. H., M. G. Pau, J. H. Custers, W. Koudstaal, S. Kostense, M. J. Havenga, D. M. Truitt, S. M. Sumida, M. G. Kishko, J. C. Arthur, B. Korioth-Schmitz, M. H. Newberg, D. A. Gorgone, M. A. Lifton, D. L. Panicali, G. J. Nabel, N. L. Letvin, and J. Goudsmit Immunogenicity of 31

32 recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti- Ad5 immunity. J Immunol 172: Barouch, D. H., S. Santra, J. E. Schmitz, M. J. Kuroda, T. M. Fu, W. Wagner, M. Bilska, A. Craiu, X. X. Zheng, G. R. Krivulka, K. Beaudry, M. A. Lifton, C. E. Nickerson, W. L. Trigona, K. Punt, D. C. Freed, L. Guan, S. Dubey, D. Casimiro, A. Simon, M. E. Davies, M. Chastain, T. B. Strom, R. S. Gelman, D. C. Montefiori, M. G. Lewis, E. A. Emini, J. W. Shiver, and N. L. Letvin Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science 290: Barouch, D. H., S. Santra, K. Tenner-Racz, P. Racz, M. J. Kuroda, J. E. Schmitz, S. S. Jackson, M. A. Lifton, D. C. Freed, H. C. Perry, M. E. Davies, J. W. Shiver, and N. L. Letvin Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF. J Immunol 168: Bellone, M., G. Iezzi, A. Martin-Fontecha, L. Rivolta, A. A. Manfredi, M. P. Protti, M. Freschi, P. Dellabona, G. Casorati, and C. Rugarli Rejection of a nonimmunogenic melanoma by vaccination with natural melanoma peptides on engineered antigen-presenting cells. J Immunol 158: Belyakov, I. M., J. D. Ahlers, B. Y. Brandwein, P. Earl, B. L. Kelsall, B. Moss, W. Strober, and J. A. Berzofsky The importance of local mucosal HIV-specific CD8(+) cytotoxic T lymphocytes for resistance to mucosal viral transmission in mice and enhancement of resistance by local administration of IL- 12. J Clin Invest 102: Belyakov, I. M., M. A. Derby, J. D. Ahlers, B. L. Kelsall, P. Earl, B. Moss, W. Strober, and J. A. Berzofsky Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge. Proc Natl Acad Sci U S A 95: Belyakov, I. M., Z. Hel, B. Kelsall, V. A. Kuznetsov, J. D. Ahlers, J. Nacsa, D. I. Watkins, T. M. Allen, A. Sette, J. Altman, R. Woodward, P. D. Markham, J. D. Clements, G. Franchini, W. Strober, and J. A. Berzofsky Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques. Nat Med 7: Benson, J., C. Chougnet, M. Robert-Guroff, D. Montefiori, P. Markham, G. Shearer, R. C. Gallo, M. Cranage, E. Paoletti, K. Limbach, D. Venzon, J. Tartaglia, and G. Franchini Recombinant vaccine-induced protection against the highly pathogenic simian immunodeficiency virus SIV(mac251): dependence on route of challenge exposure. J Virol 72: Bergquist, C., E. L. Johansson, T. Lagergard, J. Holmgren, and A. Rudin Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina. Infect Immun 65: Berkner, K. L Expression of heterologous sequences in adenoviral vectors. Curr Top Microbiol Immunol 158: Bertley, F. M., P. A. Kozlowski, S. W. Wang, J. Chappelle, J. Patel, O. Sonuyi, G. Mazzara, D. Montefiori, A. Carville, K. G. Mansfield, and A. 32

33 Aldovini Control of simian/human immunodeficiency virus viremia and disease progression after IL-2-augmented DNA-modified vaccinia virus Ankara nasal vaccination in nonhuman primates. J Immunol 172: Betts, M. R., J. F. Krowka, T. B. Kepler, M. Davidian, C. Christopherson, S. Kwok, L. Louie, J. Eron, H. Sheppard, and J. A. Frelinger Human immunodeficiency virus type 1-specific cytotoxic T lymphocyte activity is inversely correlated with HIV type 1 viral load in HIV type 1-infected long-term survivors. AIDS Res Hum Retroviruses 15: Boyer, J. D., T. M. Robinson, M. A. Kutzler, R. Parkinson, S. A. Calarota, M. K. Sidhu, K. Muthumani, M. Lewis, G. Pavlakis, B. Felber, and D. Weiner SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques. J Med Primatol 34: Boyer, J. D., T. M. Robinson, M. A. Kutzler, G. Vansant, D. A. Hokey, S. Kumar, R. Parkinson, L. Wu, M. K. Sidhu, G. N. Pavlakis, B. K. Felber, C. Brown, P. Silvera, M. G. Lewis, J. Monforte, T. Waldmann, J. Eldridge, and D. B. Weiner Protection against Simian/Human Immunodeficiency Virus 89.6P in Macaques following Co-Immunization with SHIV Antigen and IL-15- Plasmid. Proc Natl Acad Sci U S A Boyer, J. D., T. M. Robinson, P. C. Maciag, X. Peng, R. S. Johnson, G. Pavlakis, M. G. Lewis, A. Shen, R. Siliciano, C. R. Brown, D. B. Weiner, and Y. Paterson DNA prime Listeria boost induces a cellular immune response to SIV antigens in the rhesus macaque model that is capable of limited suppression of SIV239 viral replication. Virology 333: Brenchley, J. M., T. W. Schacker, L. E. Ruff, D. A. Price, J. H. Taylor, G. J. Beilman, P. L. Nguyen, A. Khoruts, M. Larson, A. T. Haase, and D. C. Douek CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 200: Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho Virologic and immunologic characterization of long-term survivors of human immunodeficiency virus type 1 infection. N Engl J Med 332: Casimiro, D. R., F. Wang, W. A. Schleif, X. Liang, Z. Q. Zhang, T. W. Tobery, M. E. Davies, A. B. McDermott, D. H. O'Connor, A. Fridman, A. Bagchi, L. G. Tussey, A. J. Bett, A. C. Finnefrock, T. M. Fu, A. Tang, K. A. Wilson, M. Chen, H. C. Perry, G. J. Heidecker, D. C. Freed, A. Carella, K. S. Punt, K. J. Sykes, L. Huang, V. I. Ausensi, M. Bachinsky, U. Sadasivan-Nair, D. I. Watkins, E. A. Emini, and J. W. Shiver Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag. J Virol 79: Catanzaro, A. T., R. A. Koup, M. Roederer, R. T. Bailer, M. E. Enama, Z. Moodie, L. Gu, J. E. Martin, L. Novik, B. K. Chakrabarti, B. T. Butman, J. G. Gall, C. R. King, C. A. Andrews, R. Sheets, P. L. Gomez, J. R. Mascola, G. J. Nabel, and B. S. Graham Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replicationdefective recombinant adenovirus vector. J Infect Dis 194:

34 31. Chakrabarti, B. K., W. P. Kong, B. Y. Wu, Z. Y. Yang, J. Friborg, X. Ling, S. R. King, D. C. Montefiori, and G. J. Nabel Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization. J Virol 76: Chen, W. C., and L. Huang Non-viral vector as vaccine carrier. Adv Genet 54: Chong, S. Y., M. A. Egan, M. A. Kutzler, S. Megati, A. Masood, V. Roopchard, D. Garcia-Hand, D. C. Montefiori, J. Quiroz, M. Rosati, E. B. Schadeck, J. D. Boyer, G. N. Pavlakis, D. B. Weiner, M. Sidhu, J. H. Eldridge, and Z. R. Israel Comparative ability of plasmid IL-12 and IL- 15 to enhance cellular and humoral immune responses elicited by a SIVgag plasmid DNA vaccine and alter disease progression following SHIV(89.6P) challenge in rhesus macaques. Vaccine 25: Clark, K. R Recent advances in recombinant adeno-associated virus vector production. Kidney Int 61 Suppl 1: Cleghorn, F., J. W. Pape, M. Schechter, C. Bartholomew, J. Sanchez, N. Jack, B. J. Metch, M. Hansen, M. Allen, H. Cao, D. C. Montefiori, G. D. Tomaras, S. Gurunathan, D. J. Eastman, R. F. do Lago, S. Jean, J. R. Lama, D. N. Lawrence, and P. F. Wright Lessons From a Multisite International Trial in the Caribbean and South America of an HIV-1 Canarypox Vaccine (ALVAC-HIV vcp1452) With or Without Boosting With MN rgp120. J Acquir Immune Defic Syndr Publish Ahead of Print. 36. Cohen, P Immunity's yin and yang. A successful vaccine must first avoid being eliminated by pre-existing immunity before it can promote a protective immune response. IAVI Report 10: Corey, L Vaccines to Induce CTL Responses to HIV in Immunocompetent Individuals: State of the Field. CROI (Colorado). 38. Croyle, M. A., N. Chirmule, Y. Zhang, and J. M. Wilson PEGylation of E1-deleted adenovirus vectors allows significant gene expression on readministration to liver. Hum Gene Ther 13: Deml, L., A. Bojak, S. Steck, M. Graf, J. Wild, R. Schirmbeck, H. Wolf, and R. Wagner Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol 75: Drosopoulos, W. C., L. F. Rezende, M. A. Wainberg, and V. R. Prasad Virtues of being faithful: can we limit the genetic variation in human immunodeficiency virus? J Mol Med 76: Earl, P. L., J. L. Americo, L. S. Wyatt, L. Anne Eller, D. C. Montefiori, R. Byrum, M. Piatak, J. D. Lifson, R. Rao Amara, H. L. Robinson, J. W. Huggins, and B. Moss Recombinant modified vaccinia virus Ankara provides durable protection against disease caused by an immunodeficiency virus as well as long-term immunity to an orthopoxvirus in a non-human primate. Virology. 42. Egan, M. A., S. Y. Chong, S. Megati, D. C. Montefiori, N. F. Rose, J. D. Boyer, M. K. Sidhu, J. Quiroz, M. Rosati, E. B. Schadeck, G. N. Pavlakis, D. B. Weiner, J. K. Rose, Z. R. Israel, S. A. Udem, and J. H. Eldridge

35 Priming with plasmid DNAs expressing interleukin-12 and simian immunodeficiency virus gag enhances the immunogenicity and efficacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus. AIDS Res Hum Retroviruses 21: Egan, M. A., S. Y. Chong, N. F. Rose, S. Megati, K. J. Lopez, E. B. Schadeck, J. E. Johnson, A. Masood, P. Piacente, R. E. Druilhet, P. W. Barras, D. L. Hasselschwert, P. Reilly, E. M. Mishkin, D. C. Montefiori, M. G. Lewis, D. K. Clarke, R. M. Hendry, P. A. Marx, J. H. Eldridge, S. A. Udem, Z. R. Israel, and J. K. Rose Immunogenicity of attenuated vesicular stomatitis virus vectors expressing HIV type 1 Env and SIV Gag proteins: comparison of intranasal and intramuscular vaccination routes. AIDS Res Hum Retroviruses 20: Evans, D. T., L. M. Chen, J. Gillis, K. C. Lin, B. Harty, G. P. Mazzara, R. O. Donis, K. G. Mansfield, J. D. Lifson, R. C. Desrosiers, J. E. Galan, and R. P. Johnson Mucosal priming of simian immunodeficiency virus-specific cytotoxic T-lymphocyte responses in rhesus macaques by the Salmonella type III secretion antigen delivery system. J Virol 77: Farina, S. F., G. P. Gao, Z. Q. Xiang, J. J. Rux, R. M. Burnett, M. R. Alvira, J. Marsh, H. C. Ertl, and J. M. Wilson Replication-defective vector based on a chimpanzee adenovirus. J Virol 75: Fitzgerald, J. C., G. P. Gao, A. Reyes-Sandoval, G. N. Pavlakis, Z. Q. Xiang, A. P. Wlazlo, W. Giles-Davis, J. M. Wilson, and H. C. Ertl A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag. J Immunol 170: Franchini, G., M. Robert-Guroff, J. Tartaglia, A. Aggarwal, A. Abimiku, J. Benson, P. Markham, K. Limbach, G. Hurteau, J. Fullen, and et al Highly attenuated HIV type 2 recombinant poxviruses, but not HIV-2 recombinant Salmonella vaccines, induce long-lasting protection in rhesus macaques. AIDS Res Hum Retroviruses 11: Frolov, I., and S. Schlesinger Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells. J Virol 68: Fynan, E. F., R. G. Webster, D. H. Fuller, J. R. Haynes, J. C. Santoro, and H. L. Robinson DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci U S A 90: Gall, J. G., R. G. Crystal, and E. Falck-Pedersen Construction and characterization of hexon-chimeric adenoviruses: specification of adenovirus serotype. J Virol 72: Gardner, J. P., I. Frolov, S. Perri, Y. Ji, M. L. MacKichan, J. zur Megede, M. Chen, B. A. Belli, D. A. Driver, S. Sherrill, C. E. Greer, G. R. Otten, S. W. Barnett, M. A. Liu, T. W. Dubensky, and J. M. Polo Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein. J Virol 74: Graham, B. S., R. A. Koup, M. Roederer, R. T. Bailer, M. E. Enama, Z. Moodie, J. E. Martin, M. M. McCluskey, B. K. Chakrabarti, L. Lamoreaux, C. A. Andrews, P. L. Gomez, J. R. Mascola, and G. J. Nabel Phase 1 35

36 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine. J Infect Dis 194: Greenland, J. R., and N. L. Letvin Chemical adjuvants for plasmid DNA vaccines. Vaccine 25: Guadalupe, M., E. Reay, S. Sankaran, T. Prindiville, J. Flamm, A. McNeil, and S. Dandekar Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 77: Gupta, S., F. Zhou, C. E. Greer, H. Legg, T. Tang, P. Luciw, J. zur Megede, S. W. Barnett, J. J. Donnelly, D. T. O'Hagan, J. M. Polo, and M. Vajdy Antibody responses against HIV in rhesus macaques following combinations of mucosal and systemic immunizations with chimeric alphavirus-based replicon particles. AIDS Res Hum Retroviruses 22: Haensler, J., C. Verdelet, V. Sanchez, Y. Girerd-Chambaz, A. Bonnin, E. Trannoy, S. Krishnan, and P. Meulien Intradermal DNA immunization by using jet-injectors in mice and monkeys. Vaccine 17: Hanke, T., R. V. Samuel, T. J. Blanchard, V. C. Neumann, T. M. Allen, J. E. Boyson, S. A. Sharpe, N. Cook, G. L. Smith, D. I. Watkins, M. P. Cranage, and A. J. McMichael Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen. J Virol 73: Harshyne, L. A., S. C. Watkins, A. Gambotto, and S. M. Barratt-Boyes Dendritic cells acquire antigens from live cells for cross-presentation to CTL. J Immunol 166: Hel, Z., J. Nacsa, B. Kelsall, W. P. Tsai, N. Letvin, R. W. Parks, E. Tryniszewska, L. Picker, M. G. Lewis, Y. Edghill-Smith, M. Moniuszko, R. Pal, L. Stevceva, J. D. Altman, T. M. Allen, D. Watkins, J. V. Torres, J. A. Berzofsky, I. M. Belyakov, W. Strober, and G. Franchini Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques. J Virol 75: Hel, Z., J. Nacsa, E. Tryniszewska, W. P. Tsai, R. W. Parks, D. C. Montefiori, B. K. Felber, J. Tartaglia, G. N. Pavlakis, and G. Franchini Containment of simian immunodeficiency virus infection in vaccinated macaques: correlation with the magnitude of virus-specific pre- and postchallenge CD4+ and CD8+ T cell responses. J Immunol 169: Hel, Z., W. P. Tsai, E. Tryniszewska, J. Nacsa, P. D. Markham, M. G. Lewis, G. N. Pavlakis, B. K. Felber, J. Tartaglia, and G. Franchini Improved vaccine protection from simian AIDS by the addition of nonstructural simian immunodeficiency virus genes. J Immunol 176: Hel, Z., D. Venzon, M. Poudyal, W. P. Tsai, L. Giuliani, R. Woodward, C. Chougnet, G. Shearer, J. D. Altman, D. Watkins, N. Bischofberger, A. Abimiku, P. Markham, J. Tartaglia, and G. Franchini Viremia control 36

37 following antiretroviral treatment and therapeutic immunization during primary SIV251 infection of macaques. Nat Med 6: Heller, L. C., and R. Heller In vivo electroporation for gene therapy. Hum Gene Ther 17: Hirao, L. A., L. Wu, A. S. Khan, A. Satishchandran, R. Draghia-Akli, and D. B. Weiner Intradermal/Subcutaneous Immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques. Vaccine JVAC_ Hofmann, C., P. Loser, G. Cichon, W. Arnold, G. W. Both, and M. Strauss Ovine adenovirus vectors overcome preexisting humoral immunity against human adenoviruses in vivo. J Virol 73: Hokey, D. A., and D. B. Weiner DNA vaccines for HIV: challenges and opportunities. Springer Semin Immunopathol 28: Holterman, L., R. Vogels, R. van der Vlugt, M. Sieuwerts, J. Grimbergen, J. Kaspers, E. Geelen, E. van der Helm, A. Lemckert, G. Gillissen, S. Verhaagh, J. Custers, D. Zuijdgeest, B. Berkhout, M. Bakker, P. Quax, J. Goudsmit, and M. Havenga Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5. J Virol 78: IAVI Vaccine Briefs. IAVI Report 11: Inaba, K., S. Turley, F. Yamaide, T. Iyoda, K. Mahnke, M. Inaba, M. Pack, M. Subklewe, B. Sauter, D. Sheff, M. Albert, N. Bhardwaj, I. Mellman, and R. M. Steinman Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J Exp Med 188: Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, L. G. Kostrikis, L. Zhang, A. S. Perelson, and D. D. Ho Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med 189: Johnson, J. E., F. Nasar, J. W. Coleman, R. E. Price, A. Javadian, K. Draper, M. Lee, P. A. Reilly, D. K. Clarke, R. M. Hendry, and S. A. Udem Neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates. Virology 360: Johnson, P. R., B. C. Schnepp, M. J. Connell, D. Rohne, S. Robinson, G. R. Krivulka, C. I. Lord, R. Zinn, D. C. Montefiori, N. L. Letvin, and K. R. Clark Novel adeno-associated virus vector vaccine restricts replication of simian immunodeficiency virus in macaques. J Virol 79: Johnston, R. E., P. R. Johnson, M. J. Connell, D. C. Montefiori, A. West, M. L. Collier, C. Cecil, R. Swanstrom, J. A. Frelinger, and N. L. Davis Vaccination of macaques with SIV immunogens delivered by Venezuelan equine encephalitis virus replicon particle vectors followed by a mucosal challenge with SIVsmE660. Vaccine 23: Jolly, D Viral vector systems for gene therapy. Cancer Gene Ther 1: Kaiser, J Gene therapy. Questions remain on cause of death in arthritis trial. Science 317:

38 76. Kantakamalakul, W., M. De Souza, C. Karnasuta, A. Brown, S. Gurunathan, D. Birx, P. Thongcharoen, and T. Taveg Enhanced sensitivity of detection of cytotoxic T lymphocyte responses to HIV type 1 proteins using an extended in vitro stimulation period for measuring effector function in volunteers enrolled in an ALVAC-HIV phase I/II prime boost vaccine trial in Thailand. AIDS Res Hum Retroviruses 20: Kaur, A., H. B. Sanford, D. Garry, S. Lang, S. A. Klumpp, D. Watanabe, R. T. Bronson, J. D. Lifson, M. Rosati, G. N. Pavlakis, B. K. Felber, D. M. Knipe, and R. C. Desrosiers Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus. Virology 357: Keulen, W., M. Nijhuis, R. Schuurman, B. Berkhout, and C. Boucher Reverse transcriptase fidelity and HIV-1 variation. Science 275:229; author reply Kong, W. P., Y. Huang, Z. Y. Yang, B. K. Chakrabarti, Z. Moodie, and G. J. Nabel Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines. J Virol 77: Kostense, S., W. Koudstaal, M. Sprangers, G. J. Weverling, G. Penders, N. Helmus, R. Vogels, M. Bakker, B. Berkhout, M. Havenga, and J. Goudsmit Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector. Aids 18: Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky, C. Farthing, and D. D. Ho Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 68: Kozlowski, P. A., S. Cu-Uvin, M. R. Neutra, and T. P. Flanigan Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women. Infect Immun 65: Kozlowski, P. A., S. B. Williams, R. M. Lynch, T. P. Flanigan, R. R. Patterson, S. Cu-Uvin, and M. R. Neutra Differential induction of mucosal and systemic antibody responses in women after nasal, rectal, or vaginal immunization: influence of the menstrual cycle. J Immunol 169: Kresge, K. J What next? IAVI Report Leitner, W. W., L. N. Hwang, M. J. deveer, A. Zhou, R. H. Silverman, B. R. Williams, T. W. Dubensky, H. Ying, and N. P. Restifo Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat Med 9: Letvin, N. L., J. R. Mascola, Y. Sun, D. A. Gorgone, A. P. Buzby, L. Xu, Z. Y. Yang, B. Chakrabarti, S. S. Rao, J. E. Schmitz, D. C. Montefiori, B. R. Barker, F. L. Bookstein, and G. J. Nabel Preserved CD4+ central memory T cells and survival in vaccinated SIV-challenged monkeys. Science 312: Li, Q., L. Duan, J. D. Estes, Z. M. Ma, T. Rourke, Y. Wang, C. Reilly, J. Carlis, C. J. Miller, and A. T. Haase Peak SIV replication in resting 38

39 memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 434: Li, S., E. Locke, J. Bruder, D. Clarke, D. L. Doolan, M. J. Havenga, A. V. Hill, P. Liljestrom, T. P. Monath, H. Y. Naim, C. Ockenhouse, D. C. Tang, K. R. Van Kampen, J. F. Viret, F. Zavala, and F. Dubovsky Viral vectors for malaria vaccine development. Vaccine 25: Liang, X., D. R. Casimiro, W. A. Schleif, F. Wang, M. E. Davies, Z. Q. Zhang, T. M. Fu, A. C. Finnefrock, L. Handt, M. P. Citron, G. Heidecker, A. Tang, M. Chen, K. A. Wilson, L. Gabryelski, M. McElhaugh, A. Carella, C. Moyer, L. Huang, S. Vitelli, D. Patel, J. Lin, E. A. Emini, and J. W. Shiver Vectored Gag and Env but not Tat show efficacy against simian-human immunodeficiency virus 89.6P challenge in Mamu-A*01-negative rhesus monkeys. J Virol 79: Lieberman, J., and F. R. Frankel Engineered Listeria monocytogenes as an AIDS vaccine. Vaccine 20: Liljestrom, P., and H. Garoff A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 9: Lin, S. W., A. S. Cun, K. Harris-McCoy, and H. C. Ertl Intramuscular rather than oral administration of replication-defective adenoviral vaccine vector induces specific CD8+ T cell responses in the gut. Vaccine 25: Liu, M. A Vaccine developments. Nat Med 4: Loessner, H., and S. Weiss Bacteria-mediated DNA transfer in gene therapy and vaccination. Expert Opin Biol Ther 4: Lu, S Combination DNA plus protein HIV vaccines. Springer Semin Immunopathol 28: Luckay, A., M. K. Sidhu, R. Kjeken, S. Megati, S. Y. Chong, V. Roopchand, D. Garcia-Hand, R. Abdullah, R. Braun, D. C. Montefiori, M. Rosati, B. K. Felber, G. N. Pavlakis, I. Mathiesen, Z. R. Israel, J. H. Eldridge, and M. A. Egan Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol 81: Mackett, M., G. L. Smith, and B. Moss Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci U S A 79: Mattapallil, J. J., D. C. Douek, A. Buckler-White, D. Montefiori, N. L. Letvin, G. J. Nabel, and M. Roederer Vaccination preserves CD4 memory T cells during acute simian immunodeficiency virus challenge. J Exp Med 203: Mattapallil, J. J., D. C. Douek, B. Hill, Y. Nishimura, M. Martin, and M. Roederer Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 434: McCoy, K., N. Tatsis, B. Korioth-Schmitz, M. O. Lasaro, S. E. Hensley, S. W. Lin, Y. Li, W. Giles-Davis, A. Cun, D. Zhou, Z. Xiang, N. L. Letvin, and H. C. Ertl Effect of preexisting immunity to adenovirus human serotype 5 antigens on the immune responses of nonhuman primates to vaccine regimens based on human- or chimpanzee-derived adenovirus vectors. J Virol 81:

40 101. McDermott, A. B., D. H. O'Connor, S. Fuenger, S. Piaskowski, S. Martin, J. Loffredo, M. Reynolds, J. Reed, J. Furlott, T. Jacoby, C. Riek, E. Dodds, K. Krebs, M. E. Davies, W. A. Schleif, D. R. Casimiro, J. W. Shiver, and D. I. Watkins Cytotoxic T-lymphocyte escape does not always explain the transient control of simian immunodeficiency virus SIVmac239 viremia in adenovirus-boosted and DNA-primed Mamu-A*01-positive rhesus macaques. J Virol 79: Mehandru, S., M. A. Poles, K. Tenner-Racz, A. Horowitz, A. Hurley, C. Hogan, D. Boden, P. Racz, and M. Markowitz Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 200: Mehandru, S., M. A. Poles, K. Tenner-Racz, V. Manuelli, P. Jean-Pierre, P. Lopez, A. Shet, A. Low, H. Mohri, D. Boden, P. Racz, and M. Markowitz Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection. J Virol 81: Molinier-Frenkel, V., R. Lengagne, F. Gaden, S. S. Hong, J. Choppin, H. Gahery-Segard, P. Boulanger, and J. G. Guillet Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response. J Virol 76: Moss, B Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci U S A 93: Mulligan, M. J., N. D. Russell, C. Celum, J. Kahn, E. Noonan, D. C. Montefiori, G. Ferrari, K. J. Weinhold, J. M. Smith, R. R. Amara, and H. L. Robinson Excellent safety and tolerability of the human immunodeficiency virus type 1 pga2/js2 plasmid DNA priming vector vaccine in HIV type 1 uninfected adults. AIDS Res Hum Retroviruses 22: Murphy, C. G., W. T. Lucas, R. E. Means, S. Czajak, C. L. Hale, J. D. Lifson, A. Kaur, R. P. Johnson, D. M. Knipe, and R. C. Desrosiers Vaccine protection against simian immunodeficiency virus by recombinant strains of herpes simplex virus. J Virol 74: Musey, L., J. Hughes, T. Schacker, T. Shea, L. Corey, and M. J. McElrath Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection. N Engl J Med 337: Neeson, P., J. Boyer, S. Kumar, M. G. Lewis, L. Mattias, R. Veazey, D. Weiner, and Y. Paterson A DNA prime-oral Listeria boost vaccine in rhesus macaques induces a SIV-specific CD8 T cell mucosal response characterized by high levels of alpha4beta7 integrin and an effector memory phenotype. Virology 354: Nwanegbo, E., E. Vardas, W. Gao, H. Whittle, H. Sun, D. Rowe, P. D. Robbins, and A. Gambotto Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States. Clin Diagn Lab Immunol 11: O'Hagan, D. T., and M. Singh Microparticles as vaccine adjuvants and delivery systems. Expert Rev Vaccines 2: Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard, J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, A. Hurley, M. 40

41 Markowitz, D. D. Ho, D. F. Nixon, and A. J. McMichael Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279: Otten, G. R., M. Schaefer, B. Doe, H. Liu, J. Z. Megede, J. Donnelly, D. Rabussay, S. Barnett, and J. B. Ulmer Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 24: Ourmanov, I., C. R. Brown, B. Moss, M. Carroll, L. Wyatt, L. Pletneva, S. Goldstein, D. Venzon, and V. M. Hirsch Comparative efficacy of recombinant modified vaccinia virus Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol and/or Env in macaques challenged with pathogenic SIV. J Virol 74: Pal, R., D. Venzon, S. Santra, V. S. Kalyanaraman, D. C. Montefiori, L. Hocker, L. Hudacik, N. Rose, J. Nacsa, Y. Edghill-Smith, M. Moniuszko, Z. Hel, I. M. Belyakov, J. A. Berzofsky, R. W. Parks, P. D. Markham, N. L. Letvin, J. Tartaglia, and G. Franchini Systemic immunization with an ALVAC-HIV-1/protein boost vaccine strategy protects rhesus macaques from CD4+ T-cell loss and reduces both systemic and mucosal simian-human immunodeficiency virus SHIVKU2 RNA levels. J Virol 80: Pal, R., S. Wang, V. S. Kalyanaraman, B. C. Nair, S. Whitney, T. Keen, L. Hocker, L. Hudacik, N. Rose, A. Cristillo, I. Mboudjeka, S. Shen, T. H. Wu- Chou, D. Montefiori, J. Mascola, S. Lu, and P. Markham Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate. J Med Primatol 34: Pinto, A. R., J. C. Fitzgerald, W. Giles-Davis, G. P. Gao, J. M. Wilson, and H. C. Ertl Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers. J Immunol 171: Rayner, J. O., S. A. Dryga, and K. I. Kamrud Alphavirus vectors and vaccination. Rev Med Virol 12: Reddy, P. S., Y. Chen, N. Idamakanti, C. Pyne, L. A. Babiuk, and S. K. Tikoo Characterization of early region 1 and pix of bovine adenovirus-3. Virology 253: Reddy, P. S., N. Idamakanti, Y. Chen, T. Whale, L. A. Babiuk, M. Mehtali, and S. K. Tikoo Replication-defective bovine adenovirus type 3 as an expression vector. J Virol 73: Roberts, A., L. Buonocore, R. Price, J. Forman, and J. K. Rose Attenuated vesicular stomatitis viruses as vaccine vectors. J Virol 73: Roberts, D. M., A. Nanda, M. J. Havenga, P. Abbink, D. M. Lynch, B. A. Ewald, J. Liu, A. R. Thorner, P. E. Swanson, D. A. Gorgone, M. A. Lifton, A. A. Lemckert, L. Holterman, B. Chen, A. Dilraj, A. Carville, K. G. Mansfield, J. Goudsmit, and D. H. Barouch Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature 441:

42 123. Robinson, H. L., D. C. Montefiori, R. P. Johnson, M. L. Kalish, S. L. Lydy, and H. M. McClure DNA priming and recombinant pox virus boosters for an AIDS vaccine. Dev Biol (Basel) 104: Robinson, H. L., and K. J. Weinhold Phase 1 clinical trials of the National Institutes of Health Vaccine Research Center HIV/AIDS candidate vaccines. J Infect Dis 194: Rock, K. L A new foreign policy: MHC class I molecules monitor the outside world. Immunol Today 17: Roy, S., P. S. Shirley, A. McClelland, and M. Kaleko Circumvention of immunity to the adenovirus major coat protein hexon. J Virol 72: Russmann, H., H. Shams, F. Poblete, Y. Fu, J. E. Galan, and R. O. Donis Delivery of epitopes by the Salmonella type III secretion system for vaccine development. Science 281: Sailaja, G., H. HogenEsch, A. North, J. Hays, and S. K. Mittal Encapsulation of recombinant adenovirus into alginate microspheres circumvents vector-specific immune response. Gene Ther 9: Schadeck, E. B., M. Sidhu, M. A. Egan, S. Y. Chong, P. Piacente, A. Masood, D. Garcia-Hand, S. Cappello, V. Roopchand, S. Megati, J. Quiroz, J. D. Boyer, B. K. Felber, G. N. Pavlakis, D. B. Weiner, J. H. Eldridge, and Z. R. Israel A dose sparing effect by plasmid encoded IL-12 adjuvant on a SIVgag-plasmid DNA vaccine in rhesus macaques. Vaccine 24: Schlesinger, S Alphavirus vectors: development and potential therapeutic applications. Expert Opin Biol Ther 1: Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, J. Ghrayeb, M. A. Forman, D. C. Montefiori, E. P. Rieber, N. L. Letvin, and K. A. Reimann Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283: Schneider, J., S. C. Gilbert, C. M. Hannan, P. Degano, E. Prieur, E. G. Sheu, M. Plebanski, and A. V. Hill Induction of CD8+ T cells using heterologous prime-boost immunisation strategies. Immunol Rev 170: Schneider, R., M. Campbell, G. Nasioulas, B. K. Felber, and G. N. Pavlakis Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation. J Virol 71: Schneider, T., H. U. Jahn, W. Schmidt, E. O. Riecken, M. Zeitz, and R. Ullrich Loss of CD4 T lymphocytes in patients infected with human immunodeficiency virus type 1 is more pronounced in the duodenal mucosa than in the peripheral blood. Berlin Diarrhea/Wasting Syndrome Study Group. Gut 37: Schnell, M. J Viral vectors as potential HIV-1 vaccines. FEMS Microbiol Lett 200: Shiver, J. W., and E. A. Emini Recent advances in the development of HIV-1 vaccines using replication-incompetent adenovirus vectors. Annu Rev Med 55:

43 137. Shiver, J. W., T. M. Fu, L. Chen, D. R. Casimiro, M. E. Davies, R. K. Evans, Z. Q. Zhang, A. J. Simon, W. L. Trigona, S. A. Dubey, L. Huang, V. A. Harris, R. S. Long, X. Liang, L. Handt, W. A. Schleif, L. Zhu, D. C. Freed, N. V. Persaud, L. Guan, K. S. Punt, A. Tang, M. Chen, K. A. Wilson, K. B. Collins, G. J. Heidecker, V. R. Fernandez, H. C. Perry, J. G. Joyce, K. M. Grimm, J. C. Cook, P. M. Keller, D. S. Kresock, H. Mach, R. D. Troutman, L. A. Isopi, D. M. Williams, Z. Xu, K. E. Bohannon, D. B. Volkin, D. C. Montefiori, A. Miura, G. R. Krivulka, M. A. Lifton, M. J. Kuroda, J. E. Schmitz, N. L. Letvin, M. J. Caulfield, A. J. Bett, R. Youil, D. C. Kaslow, and E. A. Emini Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature 415: Smith, J. M., R. R. Amara, H. M. McClure, M. Patel, S. Sharma, H. Yi, L. Chennareddi, J. G. Herndon, S. T. Butera, W. Heneine, D. L. Ellenberger, B. Parekh, P. L. Earl, L. S. Wyatt, B. Moss, and H. L. Robinson Multiprotein HIV type 1 clade B DNA/MVA vaccine: construction, safety, and immunogenicity in Macaques. AIDS Res Hum Retroviruses 20: Spira, S., M. A. Wainberg, H. Loemba, D. Turner, and B. G. Brenner Impact of clade diversity on HIV-1 virulence, antiretroviral drug sensitivity and drug resistance. J Antimicrob Chemother 51: Stevceva, L., X. Alvarez, A. A. Lackner, E. Tryniszewska, B. Kelsall, J. Nacsa, J. Tartaglia, W. Strober, and G. Franchini Both mucosal and systemic routes of immunization with the live, attenuated NYVAC/simian immunodeficiency virus SIV(gpe) recombinant vaccine result in gag-specific CD8(+) T-cell responses in mucosal tissues of macaques. J Virol 76: Stone, G. W., S. Barzee, V. Snarsky, K. Kee, C. A. Spina, X. F. Yu, and R. S. Kornbluth Multimeric soluble CD40 ligand and GITR ligand as adjuvants for human immunodeficiency virus DNA vaccines. J Virol 80: Stone, G. W., S. Barzee, V. Snarsky, C. A. Spina, J. D. Lifson, V. K. Pillai, R. R. Amara, F. Villinger, and R. S. Kornbluth Macaque multimeric soluble CD40 ligand and GITR ligand constructs are immunostimulatory molecules in vitro. Clin Vaccine Immunol 13: Sumida, S. M., D. M. Truitt, M. G. Kishko, J. C. Arthur, S. S. Jackson, D. A. Gorgone, M. A. Lifton, W. Koudstaal, M. G. Pau, S. Kostense, M. J. Havenga, J. Goudsmit, N. L. Letvin, and D. H. Barouch Neutralizing antibodies and CD8+ T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors. J Virol 78: Sun, J. Y., V. Anand-Jawa, S. Chatterjee, and K. K. Wong Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther 10: Tang, D. C., Z. Shi, and D. T. Curiel Vaccination onto bare skin. Nature 388: Taniyama, Y., K. Tachibana, K. Hiraoka, M. Aoki, S. Yamamoto, K. Matsumoto, T. Nakamura, T. Ogihara, Y. Kaneda, and R. Morishita Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 9:

44 147. Tatsis, N., S. W. Lin, K. Harris-McCoy, D. A. Garber, M. B. Feinberg, and H. C. Ertl Multiple immunizations with adenovirus and MVA vectors improve CD8(+) T cell functionality and mucosal homing. Virology Thery, C., L. Zitvogel, and S. Amigorena Exosomes: composition, biogenesis and function. Nat Rev Immunol 2: Thongcharoen, P., V. Suriyanon, R. M. Paris, C. Khamboonruang, M. S. de Souza, S. Ratto-Kim, C. Karnasuta, V. R. Polonis, L. Baglyos, R. E. Habib, S. Gurunathan, S. Barnett, A. E. Brown, D. L. Birx, J. G. McNeil, and J. H. Kim A Phase 1/2 Comparative Vaccine Trial of the Safety and Immunogenicity of a CRF01_AE (Subtype E) Candidate Vaccine: ALVAC-HIV (vcp1521) Prime With Oligomeric gp160 (92TH023/LAI-DID) or Bivalent gp120 (CM235/SF2) Boost. J Acquir Immune Defic Syndr 46: Thorner, A. R., A. A. Lemckert, J. Goudsmit, D. M. Lynch, B. A. Ewald, M. Denholtz, M. J. Havenga, and D. H. Barouch Immunogenicity of heterologous recombinant adenovirus prime-boost vaccine regimens is enhanced by circumventing vector cross-reactivity. J Virol 80: Titomirov, A. V., S. Sukharev, and E. Kistanova In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1088: Toka, F. N., C. D. Pack, and B. T. Rouse Molecular adjuvants for mucosal immunity. Immunol Rev 199: Trimble, C., C. T. Lin, C. F. Hung, S. Pai, J. Juang, L. He, M. Gillison, D. Pardoll, L. Wu, and T. C. Wu Comparison of the CD8+ T cell responses and antitumor effects generated by DNA vaccine administered through gene gun, biojector, and syringe. Vaccine 21: Tuboly, T., and E. Nagy Construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene. J Gen Virol 82: Veazey, R. S., M. DeMaria, L. V. Chalifoux, D. E. Shvetz, D. R. Pauley, H. L. Knight, M. Rosenzweig, R. P. Johnson, R. C. Desrosiers, and A. A. Lackner Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 280: Veazey, R. S., I. C. Tham, K. G. Mansfield, M. DeMaria, A. E. Forand, D. E. Shvetz, L. V. Chalifoux, P. K. Sehgal, and A. A. Lackner Identifying the target cell in primary simian immunodeficiency virus (SIV) infection: highly activated memory CD4(+) T cells are rapidly eliminated in early SIV infection in vivo. J Virol 74: Vignuzzi, M., S. Gerbaud, S. van der Werf, and N. Escriou Naked RNA immunization with replicons derived from poliovirus and Semliki Forest virus genomes for the generation of a cytotoxic T cell response against the influenza A virus nucleoprotein. J Gen Virol 82: Vogels, R., D. Zuijdgeest, R. van Rijnsoever, E. Hartkoorn, I. Damen, M. P. de Bethune, S. Kostense, G. Penders, N. Helmus, W. Koudstaal, M. Cecchini, A. Wetterwald, M. Sprangers, A. Lemckert, O. Ophorst, B. Koel, M. van Meerendonk, P. Quax, L. Panitti, J. Grimbergen, A. Bout, J. Goudsmit, and M. Havenga Replication-deficient human adenovirus type 35 vectors for 44

45 gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity. J Virol 77: Wang, S., D. J. Farfan-Arribas, S. Shen, T. H. Chou, A. Hirsch, F. He, and S. Lu Relative contributions of codon usage, promoter efficiency and leader sequence to the antigen expression and immunogenicity of HIV-1 Env DNA vaccine. Vaccine 24: Widera, G., M. Austin, D. Rabussay, C. Goldbeck, S. W. Barnett, M. Chen, L. Leung, G. R. Otten, K. Thudium, M. J. Selby, and J. B. Ulmer Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol 164: Xiang, Z., G. Gao, A. Reyes-Sandoval, C. J. Cohen, Y. Li, J. M. Bergelson, J. M. Wilson, and H. C. Ertl Novel, chimpanzee serotype 68-based adenoviral vaccine carrier for induction of antibodies to a transgene product. J Virol 76: Xiao, W., N. Chirmule, S. C. Berta, B. McCullough, G. Gao, and J. M. Wilson Gene therapy vectors based on adeno-associated virus type 1. J Virol 73: Xu, R., I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos Characterization of immune responses elicited in macaques immunized sequentially with chimeric VEE/SIN alphavirus replicon particles expressing SIVGag and/or HIVEnv and with recombinant HIVgp140Env protein. AIDS Res Hum Retroviruses 22: Yang, J. S., J. J. Kim, D. Hwang, A. Y. Choo, K. Dang, H. Maguire, S. Kudchodkar, M. P. Ramanathan, and D. B. Weiner Induction of potent Th1-type immune responses from a novel DNA vaccine for West Nile virus New York isolate (WNV-NY1999). J Infect Dis 184: Yang, S., J. W. Hodge, D. W. Grosenbach, and J. Schlom Vaccines with enhanced costimulation maintain high avidity memory CTL. J Immunol 175: Ying, H., T. Z. Zaks, R. F. Wang, K. R. Irvine, U. S. Kammula, F. M. Marincola, W. W. Leitner, and N. P. Restifo Cancer therapy using a self-replicating RNA vaccine. Nat Med 5: Youil, R., T. J. Toner, Q. Su, M. Chen, A. Tang, A. J. Bett, and D. Casimiro Hexon gene switch strategy for the generation of chimeric recombinant adenovirus. Hum Gene Ther 13: Zeira, E., A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun Femtosecond infrared laser-an efficient and safe in vivo gene delivery system for prolonged expression. Mol Ther 8: Zhang, Z., T. Schuler, M. Zupancic, S. Wietgrefe, K. A. Staskus, K. A. Reimann, T. A. Reinhart, M. Rogan, W. Cavert, C. J. Miller, R. S. Veazey, D. Notermans, S. Little, S. A. Danner, D. D. Richman, D. Havlir, J. Wong, H. L. Jordan, T. W. Schacker, P. Racz, K. Tenner-Racz, N. L. Letvin, S. Wolinsky, and A. T. Haase Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science 286:

46 170. Zhong, B., P. Tien, and H. B. Shu Innate immune responses: crosstalk of signaling and regulation of gene transcription. Virology 352: Zhou, X., P. Berglund, G. Rhodes, S. E. Parker, M. Jondal, and P. Liljestrom Self-replicating Semliki Forest virus RNA as recombinant vaccine. Vaccine 12: zur Megede, J., M. C. Chen, B. Doe, M. Schaefer, C. E. Greer, M. Selby, G. R. Otten, and S. W. Barnett Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene. J Virol 74:

47 FIGURE LEGENDS TABLE 1: The Ad5 STEP study trial design and results available to date. FIGURE 1: Vaccine induction of mucosal immunity may be important to prevent HIV- 1 dissemination. HIV-1 is primarily transmitted through the mucosal surfaces. HIV-1 specific IgA antibodies could potentially neutralize the virus, block viral adherence or entry to mucosal tissues and interfere with HIV-1 mature particle formation. CTLs specific for HIV-1 could lyse infected cells that present viral antigen via MHCI, thereby preventing their entry into the systemic compartments. FIGURE 2: Electroporation enhances both cellular (A) and humoral (B) immune responses using less amounts of DNA compared to intramuscular immunizations alone. Balb/c mice were immunized in the muscle two weeks apart with or without electroporation. The group given the intramuscular injection alone (IM) received 50ug of plasmid DNA and the electroporated group (IM/EP) 5ug or 25ug per immunization. Mice were sacrificed one week following the final immunization. (A) ELISpot with the splenocytes of mice immunized with empty plasmid (pvax) or phiv-1gag DNA (pgag). HIV-1 gag specific IFN-g secretion shown as spot forming cells (SFC) per million. Splenocytes were stimulated with 4 overlapping peptide pools spanning HIV-1 gag. (B) Antibodies against p24 HIV-1 gag as measured by ELISA. Sera were collected from immunized mice at the time of sacrifice. FIGURE 3: Electroporation increases CD4 and CD8 T cell proliferation. Rhesus macaques were immunized three times with 1.0 mg each of plasmid-encoded consensus HIV gag and rhesus IL-12 by IM immunization alone (n=2) or with electroporation (n=3). PBMCs were isolated from samples taken 2 weeks after the third immunization 47

48 and the proliferative capacity of HIV gag-specific CD4 and CD8 T cells were determined by CFSE assay. Representative dot plots for two animals from each group are shown. Percentages represent gag-specific proliferation with background proliferation (media control) subtracted. TABLE 2: A summary of the advantages, disadvantages and strategies for improvement of the reviewed HIV-1 vaccine platforms. 48

49 TABLE 1 < 1 Immunization < 2 Immunizations Viral loads a Infected Uninfected Infected Uninfected (copies/ml) Placebo ,000 Vaccinated ,000 a 8-12 weeks post diagnosis of infection 49

50 TABLE 2 Vaccine Platform Advantages Disadvantages Strategies for Improvement Poxvirus (MVA) Ability to carry large antigenic inserts Low immunogenicity in humans Remove antigenic vector sequences Can be delivered Prime-boost regimens mucosally and elicit mucosal immune responses Safe Thermostable Adenovirus Elicits best cellular and humoral immune Pre-existing immunity to most efficacious Chimp and other mammalian vectors responses in humans serotypes in humans High transduction efficiency Anti-vector immune responses develop Chimeric vectors- swap hexons Produces high titers Readministration Rare human serotypes quickly in culture difficult Easily manipulated Prime-boost regimens Antigenic inserts up to Higher doses 8kb in size Adeno-associated virus Safe Efficiently transduce Pre-existing neutralizing antibodies Prime-boost regimens New serotypes myofibers Small transgene Adjuvants capability (3.8kb) Poor immunogenicity Alphavirus Replicons Wide range of host cell types Transient expression due to replication-induced Remove antigenic vector sequences Capable of transducing cytotoxicity DCs Produce large amounts of antigenic protein Lack of pre-existing immunity in humans Induce innate and adaptive immunity Vesicular Stomatitis Virus Infects mucosal surfaces Mucosal delivery Anti-vector immune responses develop Prime-boost regimens Swap glycoproteins Low seroprevalence in humans Herpes Simplex Virus Infects mucosal surfaces Pre-existing immunity Prime-boost regimens Long-term persistence Safety concerns Bacteria (Salmonella, L. Tropism for mucosa Poor Immunogenicity in Increase antigen stability monocytogenes) Infects APCs humans Inexpensive to manufacture Vector design Prime-boost regimens DNA Safe Thermostable Poor immunogenicity in humans Genetic modifications Formulations Easy to manufacture Low antibody titer Delivery mechanisms Can boost indefinitely Molecular adjuvants 50

51 51

52