Human immunodeficiency virus antibodies and the vaccine problem

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1 Review Click here for more articles from the symposium doi: /joim Human immunodeficiency virus antibodies and the vaccine problem F. Chiodi 1 & R. A. Weiss 2 From the 1 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; and 2 Division of Infection & Immunity, University College London, London, UK Abstract. Chiodi F, Weiss RA (Karolinska Institutet, Stockholm, Sweden; University College London, London, UK). Human immunodeficiency virus antibodies and the vaccine problem (Review). J Intern Med 2014; 275: Despite the great advances made in controlling human immunodeficiency virus type 1 (HIV-1) infection with antiretroviral drug treatment, a safe and efficacious HIV vaccine has yet to be developed. Here, we discuss why clinical trials and vaccine development for HIV have so far been disappointing, with an emphasis on the lack of protective antibodies. We review approaches for developing appropriate HIV immunogens and the stimulation of long-lasting B-cell responses with antibody maturation. We conclude that candidate reagents in the pipeline for HIV vaccine development are unlikely to be particularly effective. Although the major funders of HIV vaccine research and development are placing increasing emphasis on clinical product development, a genuine breakthrough in preventing HIV infection through vaccines is more likely to come from novel immunogen research. Keywords: antibody, envelope glycoprotein, human immunodeficiency virus, vaccine. Introduction Humankind and livestock have both benefitted immeasurably from vaccines that protect against major viral disease [1, 2]. The first big success was Edward Jenner s introduction of vaccination against smallpox in Jenner named the cowpox virus vaccinia (although modern vaccinia strains might actually have an equine provenance), and immunization against infectious diseases took on the generic term vaccine from this live, attenuated virus that afforded cross-protection against smallpox. Some 90 years later, Louis Pasteur developed the first rabies vaccine from the killed extracts of infected central nervous tissue. During the 20th century, the use of vaccines against the viral diseases yellow fever, polio, mumps, measles, rubella, hepatitis B and several others has become routine, saving countless lives and deformities. Most recently, the development of vaccines against the strains of papilloma virus that cause cervical cancer in women promises to markedly reduce the occurrence of the second most common cancer in women. It is thanks to vaccination that smallpox was eventually eradicated as a naturally occurring disease in Since then, rinderpest (a morbillivirus of ruminants that is closely related to measles) has been eradicated as has poliovirus type 2. Given these successes, there is promise for the eradication of measles and poliovirus types 1 and 3. Unfortunately, not all viruses can currently be eliminated or reduced by vaccination. Progress is needed on the four serotypes of the dengue flavivirus [3]. Dengue is increasing in incidence owing to the increasing prevalence of its mosquito vector, Aedes aegypti, in urban and shantytown environments. There are two other human viruses for which vaccines are badly needed but are not yet available: hepatitis C virus and HIV-1. To these viruses, one can add the malaria parasite, Plasmodium falciparum, and the agent of TB, Mycobacterium tuberculosis, which are reviewed in accompanying articles. The successful take of a viral vaccine is usually measured by the appearance of neutralizing antibodies in the blood of the vaccinated individuals. The importance of T-cell-mediated immunity, the other arm of the immune system, for prophylactic vaccination (protection against initial infection) is debatable. T-cell immunity is certainly needed for the control of chronic, pre-existing virus infections such as the herpesviruses because immune-deficient individuals, such as persons with AIDS or 444 ª 2014 The Association for the Publication of the Journal of Internal Medicine

2 individuals deliberately immune suppressed to prevent rejection of a tissue or organ transplant, frequently develop an opportunistic resurgence of herpesviruses infection. Moreover, eliciting CD4 + T helper cells is necessary for long-lasting prophylactic humoral (antibody) immunity and the development of long-lived B memory cells with the potential to differentiate into plasma cells that release protective antibodies. Whilst humoral immunity has little effect on the progress of HIV-1 infection once established [4], broadly neutralizing antibodies show promise for preventing infection [5]. Non-neutralizing antibodies that can trigger antibody-dependent cellular responses [6] may help to control established infection, but their importance in preventing viral infections in naive subjects is less clear, as is discussed below under clinical trials. Therefore, this article will primarily focus on the induction of neutralizing antibodies to HIV-1. Immunogens and adjuvants for virus vaccines There are several ways to construct a vaccine against a specific viral disease ([1], listed in Table 1). Killed vaccines comprise whole virus particles grown in cultures or in eggs that have been rendered noninfectious by chemical agents such as paraformaldehyde. They are exemplified by the Salk polio vaccine and preserve most of the essential structures necessary to elicit immunity when inoculated into humans or animals. Liveattenuated vaccines are infectious viruses that are no longer pathogenic, and yet, they replicate in Table 1 Types of vaccines used to protect against virus infections Types of vaccines Whole killed virus Killed split virus components Live-attenuated virus Recombinant virus subunits DNA-encoding viral genes Live vectors encoding viral genes: Pox virus vectors Adenovirus vectors Adeno-associated virus vectors RNA virus vectors (Alpha-, paramyxo-, rhabdoviruses) vaccinated individuals to a degree that elicits an immune response but does not cause disease. These vaccines are exemplified by the Sabin oral polio vaccines and the measles, mumps and rubella combined vaccine (MMR). There is a risk, however, that some attenuated viruses may revert to virulence and cause disease. This has been occasionally seen with the polio vaccination, where the vaccine strain replicates in the gut. There is mutational selection for more virulent forms that are secreted and can cause mild paralysis in contacts of the vaccinated individual if they become infected. Now that wild-type polio is so rare and geographically restricted, the World Health Organization (WHO) recommends the use of a killed vaccine in areas where there is little threat of a polio epidemic. Individual components of a virus, such as an envelope or coat protein, can be effective in eliciting protective immunity. For instance, the influenza vaccine virus is grown in eggs or cell culture, inactivated and then split into component parts, of which the envelope glycoprotein, hemagglutinin, is the main protective immunogen. Recombinant proteins can replace components derived from the live virus. Immunity can be effective when components self-assemble into aggregates or virus-like particles lacking genomes; examples include the hepatitis B and cervical papilloma virus vaccines. If recombinant proteins are themselves good immunogens, then expressing the genes that encode them in the vaccinee could be a convenient and less expensive means to immunize the target population. DNA vaccines have not been licensed for other viral diseases, but there is great interest in developing them for HIV-1. Viral vectors are well tested, presumably harmless, self-replicating viruses engineered to carry genes encoding HIV proteins in addition to their own. Modified Vaccinia Ankara (MVA) and other poxvirus vectors have been intensively studied for this purpose, and a variety of vectors derived from DNA or RNA viruses are currently under investigation for the delivery and expression of HIV immunogens [2, 7]. Interestingly, Scott G Hansen and his collaborators produced a potential breakthrough in the field of HIV vaccinology. These researchers demonstrated that rhesus cytomegalovirus (RhCMV/SIV) expressing proteins from the simian immunodeficiency virus (SIV) elicited an immune response that controlled SIVmac239 infection in rhesus macaques [8]. Protection with the CMV/SIV vector was ª 2014 The Association for the Publication of the Journal of Internal Medicine 445

3 induced independently of the challenge route, and the SIV-specific CD8+ T-cell response, which was elicited by the RhCMV/SIV vector and recognized SIV-infected CD4+ T cells, remained stable over time. Moreover, no SIV RNA was detected by PCR, and no virus was isolated from the tissues of monkeys with SIV-specific T cell responses that persisted for longer time periods (over 69 weeks after infection), suggesting that immune-mediated clearance of virus infection was induced in the vaccinated animals. The mechanism by which the combined RhCMV/SIV vector elicited sustained immune control of virus replication, and clearance remains to be clarified, and it is not known whether virus-specific antibodies play a role in controlling infection. It appears that the presence of cytomegalovirus in the vector prevented the exhaustion of virus-specific T cells and led to a powerful and novel immunological method for the clearance of infected cells. All of the approaches listed in Table 1 have been tried with experimental vaccines for HIV-1. It is likely that a combination of methods, such as DNA priming followed by a protein or live-vector boost, may prove to be successful. Any particular immunogen may benefit from administration with a different adjuvant formulation. Adjuvants are not specific immunogens themselves but rather act as general stimulators of the immune system. In recent years, there have been significant advances in our understanding of the action of adjuvants derived from the discovery and characterization of toll-like receptors (TLRs) [9 11]. Different immunogens benefit from the activation of different TLRs via adjuvants that can be tailored to the type of immunogen in the vaccine. For instance, TLR4 is stimulated by DNA, and TLR9 is stimulated by recombinant protein. Judicious use of adjuvants selectively enhances the TLR response. However, the licensing of novel adjuvants for clinical use lags behind the knowledge gained from primary research, and pharmaceutical companies have been reluctant to allow testing of their proprietary adjuvants in comparison with or in combination with competing products. A live-attenuated HIV-1 vaccine based on a replication-competent form of HIV is not considered safe, even though testing of an attenuated form of SIV with a deletion in its nef gene in macaque monkeys revealed a degree of protection via an unknown mechanism [12]. Although HIV-1 has a significant potential for repair under in vivo selection, the primary reason for not using such a vaccine is that fully virulent HIV-1 itself takes 9 10 years on average to cause AIDS. Therefore, it would be very difficult to ascertain the long-term safety of an attenuated version. For example, suppose that a live-attenuated HIV-1 vaccine candidate inoculated into healthy subjects did not cause any loss of CD4 T cells and provided protection against HIV-1, but then caused neurological symptoms after a 40-year incubation period. By that point, half or more of the world s population may have been deliberately vaccinated with a socalled attenuated HIV-1 strain, clearly an unacceptable situation. Animal models One of the hurdles in HIV vaccine research and development is a lack of suitable animal models. HIV is a lentivirus, indicating that the virus causes a slowly progressive disease. Bj orn Sigurdsson first coined this term in the 1950s for Maedi-Visna virus in sheep. Other lentiviruses occur in cattle, goats, cats, horses and primates. Many species of African monkeys and apes harbour lentiviruses related to HIV-1 or to HIV-2 that seldom cause disease in the natural host but can cause AIDS in Asian macaque species, although macaques are not a natural reservoir. Whilst all known lentiviruses infect macrophages, only feline and primate lentiviruses also target T lymphocytes. It is notable that macrophage-tropic lentiviruses lead to progressive wasting and neurological symptoms similar to those seen in AIDS [13]. Cells from the myeloid lineage, such as macrophages and dendritic cells, are also targeted by HIV-1 [14]. The only lentiviral disease from which animals occasionally recover spontaneously is equine infectious anaemia in horses and donkeys. The recovery appears to correlate with the appearance of broadly neutralizing antibodies, bnabs, which has further focused research interest on humoral immunity. Unfortunately, efficacious protective vaccines have not been developed for any of these lentiviral animal diseases [15]. Human immunodeficiency virus-1 itself originated from the chimpanzee and possibly the gorilla [16]. Chimpanzees are the only animals that have been successfully infected experimentally by HIV-1, but they can no longer be used for vaccine research because they are a critically endangered species. Also HIV-1 and the chimpanzee precursor, SIVcpz, are less virulent in apes. Thus, whilst protection 446 ª 2014 The Association for the Publication of the Journal of Internal Medicine

4 against infection could be tested, it would be more difficult to determine protection against disease. The construction of a chimeric, recombinant macaque SIV in which the SIV envelope gene has been replaced with the HIV-1 envelope gene has provided a useful animal model for humoral immunity to HIV-1. Such hybrid viruses are called SHIV, and they can replicate in macaques upon experimental infection or mucosal application. Exploiting SHIVs with envelopes derived from various HIV-1 subtypes allows the testing of protection against infection either by antibodies administered passively [17 19] or following immunization with antigens and adjuvants [20, 21]. gp120 gp41 Quaternary V1V2 cluster Virus MPER cluster Conserved glycan/ glycopeptide cluster CD4 binding site cluster Small animals such as mice, guinea pigs and rabbits will continue to be useful guides for immunogens capable of inducing neutralizing antibodies. Although there are always caveats regarding immune responses in different species, such experiments are relatively inexpensive and worthwhile prior to the testing of novel immunogens in nonhuman primates and humans. Humanized mice are genetically immunodeficient mice with transplanted human immune cells or tissues that can sustain HIV-1 replication [22]. Limited immunogen tests can be carried out with these mice. The expression of neutralizing antibodies in vivo via gene transfer as a therapeutic agent has also been tested in this type of mouse [23]. Obstacles to HIV vaccine development based on envelope glycoproteins Protective B-cell immunity to HIV-1 is directed to the envelope glycoproteins gp120 and gp41. These proteins are presented on the surface of the viral particle (virion) as a small number of trimeric spikes (Fig. 1). Envelope variability The pandemic strain of HIV-1 is known as group M. During its spread around the world over the past century, HIV-1 M has diverged into multiple genetic subtypes (A K) that have also generated circulating recombinant forms (CRF) or variants [24]. This high degree of variation is reflected in antigenic diversity, especially of envelope glycoproteins. There is strong evolutionary pressure for HIV-1 to escape immune attack by mutation [25, 26]. Even for well-understood infections such as the influenza A virus, the WHO must determine each year which vaccine strains match the Fig. 1 Schematic diagram of the envelope glycoprotein complex of human immunodeficiency virus-1. The complex is shown as a trimeric structure in the lipid bilayer of the virus envelope. The gp41 glycoproteins span the lipid bilayer whilst the gp120 glycoproteins are attached noncovalently to the gp41 molecules. The major broadly neutralization-sensitive epitopes on gp41 and on gp120 are labelled. prevalent strains most closely. Unlike influenza virus, many different strains, subtypes and CRFs are present in different regions of the world in the ongoing HIV-1 pandemic. Whilst there have been recent advances in identifying broadly neutralizing antibodies to multiple strains of both HIV and influenza A [5], there is still a formidable obstacle to the development of a universal vaccine that would offer protection against the majority of HIV strains. Experimental immunogens based on the HIV-1 envelope can induce antibodies that at least weakly neutralize the homologous strain, but this result is not sufficient for the protection of the human population at large. Immunodominant and cryptic epitopes on the envelope Approximately 50% of the molecular weight of the HIV-1 envelope glycoprotein consists of carbohydrate groups that cover the proteinaceous parts of the envelope such as a glycan shield. Whilst certain carbohydrate moieties are targets themselves for neutralizing antibodies or contribute to complex tertiary epitopes, they primarily act to obscure the known neutralization-sensitive epitopes [27]. In addition, other non-neutralizing epitopes tend to dominate the natural B-cell responses to envelope antigens and do not afford protection. This problem might be partially solved if immunogens could be designed that focus the dominant antibody ª 2014 The Association for the Publication of the Journal of Internal Medicine 447

5 Maintenance of serological memory through memory B cells and long-lived plasma cells Lymph node Blood Bone marrow DC F T FH P L S Germinal center GCB P P S P L P L S P L B M Fig. 2 Maintenance of serological memory through memory B cells and long-lived plasma cells. Serological memory can be defined as a persistent pool of protective antibodies against a specific antigen previously met in life, through vaccination or natural infection. Two types of B cells are behind maintenance of long-term serological memory, memory B cells residing in spleen and long-lived plasma cells which reside in the spleen. These cells are generated as result of the complex interactions taking place in the germinal centre (GC) between GC B cells, T follicular helper (TFH) cells and follicular dendritic cells (FDCs); as result of these interactions mediated by several molecular contacts, GC B cells differentiate into memory B cells and plasma cells. Plasma cells migrate to the bone marrow where they become long-lived plasma cells through the survival signals provided from stromal cells. It is still poorly studied whether human immunodeficiency virus-1 impairs any or all these processes. response on relatively conserved neutralizing epitopes [9, 26, 28]. Lessons learnt from natural B-cell immunity to HIV-1 In general, HIV-infected subjects produce low titres of neutralizing antibodies that have rather narrow specificity [29]. However, these antibodies may afford some protection because longitudinal studies of infected people indicate that the virus mutates to escape sensitivity to neutralization during the course of infection [25, 30]. Each time HIV-1 is transmitted from one person to another, there is a severe bottleneck of variability, and often a single transmitted viral strain or founder virus propagates in the new host. This virus replicates rapidly and begins to diversify in sequence when the immune response develops a few weeks after infection [31]. Ongoing diversification means that the virus keeps ahead of the antibody response in a form of antigenic drift within the infected individual. Nonetheless, certain individuals have been identified as elite controllers of HIV infection, some of whom are elite immune responders who make strong cellmediated responses or strong neutralizing antibody responses. These rare patients have provided a great deal of insight into what will be needed to engender protective immunity in vaccines. In the past five years, epitopes on the gp41 and gp120 envelope glycoproteins that are targeted by protective antibodies have become well defined through the isolation and analysis of monoclonal bnabs from these elite neutralizing patients [28, 32]. In parallel, a better understanding of the structure and functions of the envelope trimer (Fig. 1) is helping to pinpoint which epitopes are needed for protective antibody immunity [33 35]. However, there is still controversy over the correct depiction of the three-dimensional structure of the functional envelope spike and its flexible conformation on the virion. The binding site on gp120 for CD4 could be one Achilles heel of the HIV-1 envelope were antibodies able to gain access to this epitope and block the docking of HIV-1 with the CD4 cell surface receptor [32, 33, 36]. Once the virus binds to CD4, conformational changes in the envelope take place that open up access to the CCR5 co-receptor binding site, and this too may be a suitable target for bnabs. Another target is at the tip of variable loop 3 (V3 loop) [37], but this epitope has less breadth because this site is hidden beneath the surface in many HIV-1 strains until after the CD4-receptor interaction has taken place. A site at the membrane proximal envelope region (MPER) on gp41 has been known 448 ª 2014 The Association for the Publication of the Journal of Internal Medicine

6 for 20 years and has recently come back to prominence through newer, more potent nmabs [38]. Carbohydrates form part of the neutralization epitopes and, in the case of 2G12 nmab, the entire epitope. Perhaps the most interesting of the newly revealed epitopes is one situated at the base of the V1 V2 loop interface [39], but it is unlikely to be readily mimicked in a designed immunogen because it is a tertiary conformational epitope. Thus, it appears that there are a limited number of epitopes on HIV-1 envelope glycoproteins that are recognized by potent and broadly neutralizing antibodies and thus could be exploited in vaccine design [40]. These neutralization-sensitive epitopes have been modelled in structural studies of crystallized glycoproteins complexed with fragments of bnmabs, and the precise structures of these epitopes are reviewed elsewhere [33]. However, some recent papers have provided more detail on the conformational epitopes of highly potent bnabs [34, 35]. The problem remains how to translate this new knowledge of cross-specific antigens into immunogens that will induce powerful, protective responses following vaccination. Inducing long-term, protective humoral immunity to HIV: learning from immunopathology The few trials conducted with candidate HIV-1 vaccines (discussed below) showed that it is difficult to elicit high titres of HIV-1-neutralizing antibodies in vaccinated individuals, and the low levels of antibodies mounted against HIV-1 vaccines were short lived. Accordingly, these results showed that HIV vaccines have thus far failed to induce serological memory, an important aspect of successful vaccines [10]. Serological memory is defined as a persistent pool of protective antibodies against a specific antigen previously encountered through vaccination or natural infection. It has been shown that for many antigens presented to the immune system during vaccination or natural infection, measles as an example, the level of protective antibodies remain constant for an extended period of time, up to the full lifespan of the individual [41]. Several hypotheses have been proposed to explain the immunological mechanisms behind the maintenance of long-term serological memory. For example, memory B cells (MBCs) specific for one antigen may be activated in a polyclonal manner by other pathogens, or long-lived plasma cells (PCs) may persist for the individual s full lifespan. A short summary of the B-cell differentiation process may help the reader to follow events leading to the establishment of serological memory. B cells develop in the bone marrow and exit this organ as immature transitional B cells. These cells are present at low levels in the peripheral blood of healthy individuals, but increase during HIV-1 infection [42]. Following the elimination of autoreactive B cells via negative selection in the spleen, naive B cells migrate to T-cell-rich areas in the lymphoid tissue in response to exogenous antigens, thus initiating the formation of a germinal centre (GC). In GCs, B cells mature, expand and undergo further selection in response to antigens. Mature activated B cells will differentiate into MBCs or PCs, which leave the lymphoid tissue and travel to their niches in the bone marrow (Fig. 2). Our studies on HIV-1-infected children and adults have shown that blood MBCs are reduced in number during HIV-1 infection and that the decline in MBCs correlated with a reduction in Ab titres against measles, tetanus and pneumococcus [43, 44]. Initiation of antiretroviral therapy (ART) within the first year of life permits the normal development and maintenance of MBCs in HIV-1- infected children. As our capacity to characterize different subpopulations of memory B cells has improved through the identification of several B cell lineages and differentiating antigens, it has become clear that tissue-like memory (TLM) B cells possess the memory of HIV-1 antigens in infected patients [42, 45]. These cells have been called TLM B cells in view of their similarities to tonsilar B cells, including the expression of the inhibitory receptor, Fc-receptor-like-4 (FCRL4). During HIV-1 infection, these cells increase in number, and it has been shown that TLM B cells have an exhausted phenotype and are more susceptible to Fas-mediated apoptosis [45]. It is possible that the expression of inhibitory receptors on the surface of TLM B cells during HIV-1 infection may engage a specific pathway that leads to the inhibition of B cell proliferation [46] and antibody production. Interestingly, post-transcriptional FCRL4 gene silencing triggered with sirna in B cells from HIV-1-infected individuals caused increased levels of HIV-1-specific, antibody-secreting cells in vitro [46]. In conclusion, if HIV-1-neutralizing antibodies are produced from TLM B cells and these cells have an exhausted phenotype accompanied by the expression of negative regulators of antibody production, it is probable that only low levels of ª 2014 The Association for the Publication of the Journal of Internal Medicine 449

7 neutralizing antibodies would be found during HIV-1 infection. One question is whether FCRL4 silencing in vivo during HIV-1 infection with immunotherapy or manipulation of protein expression could result in a lower level of TLM B cell exhaustion and increased production of HIV-1-neutralizing antibodies. Very few reports have analysed the contribution of long-lived PCs to the maintenance of HIV-1 antibody responses in infected patients. It has, however, been shown that treatment of HIV-1 infection with Rituximab leads to reduced production of HIV-1-neutralizing antibodies, suggesting that MBCs are the relevant compartment for the maintenance of HIV-1 antibody titres [47]. Rituximab is an anti-cd20 monoclonal antibody that depletes MBs but not PCs lacking CD20 expression. This agent is used as an effective immunotherapy for the treatment of tumours from the B cell lineage. The increase in viral load following depletion of MBCs by Rituximab treatment also provides evidence that HIV-1-neutralizing antibodies may play a role in the control of viremia in vivo. To generate an efficient antibody response upon immunization, antigen-stimulated B cells go through class switch recombination (CSR) and affinity maturation following contact with T cells in the GCs. This process is driven by somatic hypermutation (SHM) of Ig variable genes [48], all of which is mediated by the activity of induced cytidine deaminase. These mutations are focused in the CDR region of the antibody, a region that typically contacts the antigen. Interestingly, the heavy-chain genes of the new generation of broad and potent HIV-neutralizing antibodies were shown to carry a very large number of V H mutations (>80) relative to human immunoglobulin G (10 20 mutations) [49]. Whether special molecular mechanisms operate in the GCs to generate this high number of mutations in HIV-1-neutralizing antibodies is not completely understood, but it is clear that the high number of V H mutations confers these antibodies with strong neutralizing capability. In previous discussions on the mechanisms that lead to these special genotypes for human antibodies with strong and broad HIV-1- neutralizing activity, very little attention was paid to the cell type, the TLM cells that are likely to produce these antibodies in vivo during HIV-1 infection and their exhaustion status. Assuming that TLM cells are the memory cells that carry serological memory to HIV-1 antibodies, it is possible that resetting the B-cell-activation capabilities of TLMs through the silencing of inhibitory receptors such as FCRL4 may lead to faster production of HIV-1-neutralizing antibodies in vivo. Another interesting approach that led to the ameliorated production of SIV-specific antibodies in SIV-infected macaques is blockage of the PD-1 pathway with PD-1 antibodies [50]. In the context of chronic viral infections, PD-1 has been linked to an exhausted T-cell phenotype with declined effector functions [51]. In the context of B cells and antibodies, PD-1 blockage resulted in a successful therapeutic strategy that enhanced the survival and proliferation of memory B cells and the production of SIV-specific and nonspecific antibodies [50]. All the approaches described above that aimed to improve B-cell responses during SIV and HIV infections and promote production of neutralizing antibodies by the infected hosts have an important caveat; as both FCRL4 and PD-1 molecules have a regulatory role in delineating the fate of B cells exposed to antigens within the GCs, caution should be taken not to tip the process of antibody production towards an autoimmune phenotype. To replace ART therapy with immunotherapy for HIV- 1-infected patients, it would probably be safer to concentrate on the development of already isolated neutralizing HIV antibodies with high potency and broad spectrum. This process of innovation is proceeding rapidly [52], and if cocktails of highly effective HIV-neutralizing antibodies are not too expensive, their administration to infected patients for the purpose of controlling viral dissemination may become a reality in the short term. Therefore, we should not abandon research into the possibility of inducing HIV-1-neutralizing antibodies in vivo through vaccination, but, as we will comment on in the following sections, innovative approaches are needed in this field. Previous work [53] has shown that broadly neutralizing antibodies against hepatitis C virus (HCV) were induced by a recombinant measles virus expressing structural HCV protein, and this approach may also be useful in the field of HIV-1 humoral immunity, as discussed below. Reverse vaccinology A promising area of vaccine development is called reverse vaccinology, whereby knowledge of the molecular structure of antigens is built into unnatural immunogens to elicit neutralizing epitopes [2, 450 ª 2014 The Association for the Publication of the Journal of Internal Medicine

8 54, 55]. The major envelope immunogens tested experimentally to date are recombinant, trimeric soluble gp140 molecules containing the extracellular portion of gp41 coupled with gp120 (rgp140). They are immunogenic when administered with effective adjuvants, but overall have not elicited strong neutralizing responses. This may be because they do not assemble into native conformations [28, 56, 57]. However, some of the neutralization sites, such as the CD4 binding site, are well preserved in recombinant gp140 and gp120, whilst others, such as the V1/V2 site recognized by bnmab PG9, are not. As natural infection seldom induces broadly neutralizing antibodies, there may be additional problems of immunogenicity in addition to mimicking the native trimer. Furthermore, most bnmabs are hypermutated from germline Ig sequences and will require repeated immunizations with candidate vaccines [58]. On the whole, monoclonal bnabs have not been identified in animals experimentally inoculated with rgp140. One exception, however, was the isolation of broad and potent monoclonal antibodies from llamas [40, 59]. It is not clear whether this was due to special properties of single, heavy-chain llama antibodies, or whether the immunogen (which was a mixture of rgp140s from subtype A and C strains of HIV) was particularly effective. Seeking constructs that elicit neutralizing Abs more effective than those identified in natural infection is an aim of reverse vaccinology. There is optimism that reverse vaccinology can be used to design constructs that expose the neutralization sites on native trimers more effectively [57]. In addition, artificial scaffolds can be used to present neutralization epitopes in a way that will promote the required antibody response [60]. However, until the controversy surrounding the native structure of the functional envelope spike or trimer is resolved [56], debate will continue on how best to model candidate vaccines. Recently discovered conformation-dependent bnmabs will be useful for testing the binding to empirically or rationally designed immunogens alongside structural studies. Immunization of small animals should help guide the immunogenicity of reverse-designed vaccine candidates before taking selected candidates through to nonhuman-primate immunization studies and phase I human trials. Clinical trials of candidate HIV-1 vaccines and non-neutralizing antibodies During the past decade, a small number of putative vaccines have been taken into phase II or phase III clinical trials to test efficacy in populations at risk for HIV-1 infection [28]. Four of the major trials are listed in Table 2. The VAX 003 (in USA) and 004 (in Thailand) trials tested the immunogenicity and efficacy of a recombinant monomeric gp120 envelope antigen derived from the most prevalent HIV-1 subtype in each country. The Merck 023 STEP international trial tested the effect of an Adenovirus 5 vector expressing the Gag, Pol and Nef proteins but not the envelope. It resulted in no protection and a questionable increase in HIV-1 infections for the vaccine compared to the placebo. However, this increase may have been confounded by an imbalance in the ratio of circumcised males in the two arms. The RV144 trial in Thailand was a large efficacy trial containing at least 8000 HIV-negative volunteers in each arm. The HVTN 505 trial in USA used a DNA priming immunization followed by an adenovirus vector boost expressing the Gag, Pol, Nef and Env proteins. However, this trial was halted prematurely in April 2013 by the data safety and monitoring committee because of a lack of demonstrated efficacy and a suspected increase in HIV-1 infection. Only the RV144 trial conducted amongst at risk volunteers in Thailand gave any sign of protection, Table 2 Phase II and III human vaccine trials for human immunodeficiency virus-1 Vaccine trial Year completed Approx. no. of volunteers Immunogens Targeted responses Efficacy of protection VAX rgp120 protein CD4+ Ab None Merck Ad5 gag/pol/nef CD8+ CD4 None RV ALVAC + rgp120 CD8+ CD4 31% NS HVTN DNA + Ad5 Gag/pol/nef/ env CD8+ CD4+ Ab None ª 2014 The Association for the Publication of the Journal of Internal Medicine 451

9 but the estimated degree of protection (31%) was short lived (<6 months) and did not reach statistical significance [61]. Detailed analysis of the immune responses due to vaccines compared to placebo controls indicated that non-neutralizing antibodies might have played a role in the supposed protection [62]. A number of Phase I/II trials (immunogenicity with or without efficacy) are under consideration. This is a rapidly changing field and one may reference the various HIV vaccine information sites to see which of the trials will proceed ( html; Disappointing findings from HIV vaccine trials thus far and the possible correlates of protection in RV144 [62] raises the question of whether we have been too focused on neutralization epitopes in the humoral aspects of HIV-1 vaccine research. Nonneutralizing epitopes of HIV expressed on the surface of infected cells may permit the destruction of these cells via antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent, cellmediated virus inhibition (ADCVI) [6] or lysis of HIV particles via complement-mediated attack on the virus envelope [63]. Yet, there is little evidence of these phenomena in vaccinees or in elite controllers of natural infection. If ADCC or virolysis via non-neutralizing antibodies were important correlates of protection, one would expect many more infected persons to control HIV infection because these antibodies occur more frequently, and we would see fewer mutations that lead to escape from neutralizing antibodies. A potential concern for immunization is that inappropriate humoral immune responses may increase the risk of infection. Certain non-neutralizing antibodies can enhance the efficiency of infection by promoting the attachment of non-neutralized viruses to the Fc receptors of target cells, as has been seen in dengue virus infection in the presence of heterologous antibodies to other serotypes [3]. In the case of HIV-1, antibody-dependent enhancement via Fc receptors is being examined [64]. Complement-mediated enhancement of infection has also been observed with non-neutralizing anti- HIV antibodies, although high-titre, neutralizing antibodies mask this effect [65]. The importance of mucosal immunity As the vast majority of new HIV infections occur via sexual transmission, an efficacious vaccine will need to protect the vaccinated individual from mucosal infection. The mucosa is the site of IgA secretion, and there is growing evidence that local production of IgA in the genital mucosa in HIVexposed people who have not sero-converted to systemic production of anti-hiv IgG has a protective effect against sexual transmission [66]. A small proportion of female sex workers who are frequently exposed to HIV-1 are resistant to infection, and this phenomenon is associated with IgA antibodies and possibly also with cell-mediated immunity [67]. Construction of an IgA2 isotype of an MPER gp41 mab derived from the IgG mab 2F5 was strongly neutralizing [68] and revealed that the constant region of the IgA heavy-chain contributed to overall protection [69]. Thus, neutralizing IgA antibodies may play an important role in reducing the likelihood of heterosexual transmission of HIV-1. However, a recent study of passive mucosal transfer of human monoclonal IgA antibodies to gp120 followed by mucosal challenge of SHIV demonstrated that whilst the IgA1 isoform was highly protective, the IgA2 isoform was less effective than IgG1 [19]. This observation was in contrast to the effectiveness of IgA2 reported for the MPER region of gp41 [68]. Therefore, careful analysis of the respective roles of the two isoforms of mucosal IgA merits further study. Moreover, before pinning all hopes on IgA humoral immunity in HIV-1 vaccine design, one should consider that the remarkable success of mucosal immunity in women against the human papilloma viruses associated with cervical cancer has been achieved by systemic immunization that produced primarily IgG antibodies, which are also secreted or exuded into the genital mucosa. Passive delivery of neutralizing antibodies to the mucosal surface may also present an alternative to antiretroviral drugs in vaginal microbicides [19, 32, 70 72]. In addition, novel approaches to mucosal antibody delivery are attracting interest. For example, recombinant antibodies can be produced in the vagina by commensal lactobacilli [73], and neutralizing llama antibody fragments that penetrate the mucus well can be steadily released from vaginal gels or rings [74]. Prophylactic and therapeutic antibody-based protection Recent studies using the SHIV macaque model have shown that passive introduction of antibodies not only prevents acquisition of mucosal infection 452 ª 2014 The Association for the Publication of the Journal of Internal Medicine

10 [18, 19], but can also significantly reduce viral load in the plasma in response to established infection [75, 76]. A problem with this approach is that protection wanes as the antibody titre falls. To maintain antibody levels, researchers have delivered adenovirus-associated viral vectors carrying a gene that expresses HIV-1-neutralizing antibody in humanized mice [23]. The Foundation for Vaccine Research recently convened a workshop to discuss the practicality of gene delivery of protective antibodies to HIV-1 [77]. Researchers at this workshop raised the possibility not only of therapeutic vaccines (immunotherapy) based on neutralizing antibodies, but also of immunoprophylaxis to protect against HIV infection. Which way forward in the search for an HIV-1 vaccine? We doubt that the overall HIV vaccine endeavour has identified the best method to increase protection for the millions of people at risk of acquiring HIV-1 in the coming years. The RV144 clinical trial has had an overriding effect on the policy of funding agencies for HIV vaccine research and development. Governmental and charitable foundations have poured enormous support into analysis of the results and future design based on this partial and dubious success. If funds were unlimited, one would have no criticism of this policy, but unfortunately, this switch in emphasis has come at the cost of support for innovative research into novel immunogens. Thus, there is a dilemma over future directions for HIV vaccine research and development. Some influential funding agencies appear to believe that the short-lived success in the RV144 trial should take precedence over the discovery phase of vaccine research. They appear to be interested only in products already in the production pipeline for large-scale phase III trials. They also have argued that basic research scientists are purists who do not wish to take anything forward into the clinic that does not guarantee a high level of protection that the best must not be the enemy of the good and that researchers have had long enough to come up with better candidates. In contrast, many HIV researchers regard this path as most likely to lead to failure due to the garbage in, garbage out argument, that is the quality of current vaccine candidates do not merit enormously expensive phase III trials and that more failures may lead to fatigue amongst funders for vaccines as a means of controlling HIV. They argue that it appears reckless to grasp at straws such as the RV144 results to such an overwhelming extent. The premature termination of the HVTN505 trial in April 2013 (which included env immunization, Table 2), due to fears that, such as Merck 023, the vaccine may be doing more harm than good by increasing the risk of infection has served to strengthen the sense of frustration of both sides of the debate. We believe that there is much to be gained in the long-term fight against HIV and AIDS by supporting more immunogen discovery research and a greater effort to determine what it takes to induce long-lasting protective antibody production. This approach should be coupled with testing candidate vaccines in nonhuman primates and small phase I trials of immunogenicity in low-risk human volunteers. Of course, even a small phase I trial in humans requires moderately expensive production of high-grade GMP immunogens formulated with safe adjuvants, but it is still inexpensive compared to the infrastructure of phase III trials. A lack of serious interest in such an approach might delay eventual success in the production of safe and efficacious vaccines against HIV-1. Conflict of interest statement The authors have no conflicts of interest. RAW declares that he chairs the Scientific Advisory Committee of the International AIDS Vaccine Initiative and is a member of the board of the KwaZulu-Natal Research Institute for Tuberculosis and HIV. Acknowledgements We thank Mattias Karlen for drawing the figures. The authors research has been supported by grants from the European Union, Bill & Melinda Gates Foundation, Swedish Medical Research Council and UK Medical Research Council. The views expressed in this article are the author s alone. References 1 Plotkin S, Orenstein W, Offit PA. Vaccines 2012; Expert Consult Basic Books. 2 Koff WC, Burton DR, Johnson PR et al. Accelerating next-generation vaccine development for global disease prevention. Science 2013; 340: Heinz FX, Stiasny K. Flaviviruses and flavivirus vaccines. Vaccine 2012; 30: ª 2014 The Association for the Publication of the Journal of Internal Medicine 453

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