Local control of repeat dose rectal challenges in DNA/MVA vaccinated. macaques protected against a 1 st series of SIV challenges

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1 JVI Accepts, published online ahead of print on 26 February 2014 J. Virol. doi: /jvi Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Local control of repeat dose rectal challenges in DNA/MVA vaccinated macaques protected against a 1 st series of SIV challenges 3 4 Sunil Kannanganant 1, Salaija Gangadhara 1, Lilin Lai 2, Benton Lawson 1, Pamela A Kozlowski 3, Harriet L Robinson 4, Rama Rao Amara 1,5,# 1 Division of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA 2 The Hope Clinic of the Emory Vaccine Center, Division of Infectious Diseases, Emory University, Decatur, GA, USA 3 Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA, USA 4 GeoVax, Inc., Smyrna, GA, USA 5 Department of Microbiology and Immunology, Emory University, Atlanta, GA Running title: Local control of rectal SIV challenges #To whom correspondence should be addressed: Rama Rao Amara, Phone: (404) ; FAX: (404) ; ramara@emory.edu

2 Abstract: Here we report the results of a late boost and three additional series of simian immunodeficiency virus (SIV) challenges in seven DNA/MVA vaccinated macaques who resisted a first series of rectal challenges. During 29 additional challenges delivered over 2.3 years, all animals became infected. However, 13 blips of virus in six rhesus and anamnestic Env-specific rectal IgA responses in three of the six suggested that local control of infections was occurring during the serial challenge In 2011 we reported on the prevention of infection by 12 repeated SIVE660 challenges in 5 rhesus macaques receiving a granulocyte-macrophage colony stimulating factor (GM-CSF) adjuvanted DNA/MVA vaccine and in 2 animals receiving a non-adjuvanted vaccine(6). Animals had been vaccinated with two i.m. inoculations of 3 mg of a SIV239 Gag, Pol, Env expressing DNA vaccine (D), or two inoculations of the same vaccine that co-expressed GM-CSF (Dg), followed by two i.m. boosts with 1x10 8 plaque forming units (PFU) of a SIV239 Gag, Pol, Env expressing MVA vaccine (M) in regimens designated DDMM or DgDgMM (6). The 1 st series of SIVE660 challenges was undertaken 6 months after the initial series of immunizations. Here we monitor the protected animals for one year for the presence of an occult infection, give a late MVA boost, wait 6 months and then subject the animals to a 2 nd series of 12 rectal SIVE660 challenges followed by 3 rd and 4 th series of rectal SIV251 challenges (Fig. 1A). The goals of the study were to learn more about the effects of late boosts on vaccine responses and the longevity of vaccine-mediated protection. Impressively, 6 out of the 7 animals were protected from the 2 nd series of SIVE660 challenges (Fig. 1B). The animal in the DgDgMMM group with both the lowest and a falling avidity for Env-specific Ab (Fig. 2B), was infected at the 10 th challenge (Fig. 1C). The first series of SIV251 challenges was initiated with a dose of 120 TCID 50. When the 5 unvaccinated controls were slow to become infected (one infection in 5 challenges), the challenge dose was increased to 650 TCID 50. The higher dose infected the 4 uninfected

3 45 46 controls within 4 challenges. All 6 of the vaccinated animals became infected with the last animal becoming infected at challenge Figure 2 shows the patterns of immune responses through the initial series of immunizations, the first series of challenges, the one year observation period before the late MVA boost, the third late MVA boost and six months past the late MVA boost. Consistent with macaques not becoming infected, animals that were protected during the 1 st series of challenges showed no changes in the magnitudes of their SIV-specific serum IgG or rectal IgA responses during the 1 st series of challenges or during the one year observation period prior to the late boost (Fig. 2B and 2C). The magnitudes of Ab and T cell responses expanded and contracted with MVA boosts while the quality of the Ab, as measured by avidity, tended to increase with boosts (Fig. 2). Following the late MVA boost, the magnitudes of elicited Ab, CD4 and CD8 T cell responses expanded to peak values similar to those observed in the first series of immunizations (Fig. 2 B-E). The only parameter to show a trend for a significant increase was the avidity of the Env-specific IgG response, which rose in 6 of the 7 rhesus from indices in the mid 50s post the first series of immunizations to indices in the mid 60s post the late MVA boost (p=0.06) (Fig. 2A). The contractions of the serum IgG, rectal IgA and T cell responses were overall similar following the 1 st series of immunizations and the late MVA boost. The one exception was the contraction of CD4 T cells which was less acute after the late MVA boost than after the initial series of immunizations (p=0.05) (Fig. 2D). Most of the contraction of responses occurred in the 1 st 12 weeks post immunization Throughout the 2 nd series of E660 challenges and the two series of SIV251 challenges, transient blips of RNA (<1000 copies, verified by an independent repeat assays) were detected in the 6 serially challenged animals to enter the 3 rd and 4 th series of challenges. Thirteen of the 14 blips were not associated with an anamnestic Env-specific IgG response in serum and are considered to not have established systemic

4 infections (Fig 3A and 3B). Three of the animals with blips had significant expansions of Env-specific IgA but not IgG in rectal secretions (rhesus RLe11, RWg11 and RFm11, Fig. 3C). This would be consistent with a rectally contained infection stimulating a local IgA response without stimulating a local IgG response (3). For one animal, RKk11, the final blip of SIV251 RNA was associated with an anamnestic serum IgG response, but the failure to establish viremia (Fig. 3). This animal was considered infected and represented the last animal to become infected. Post infection levels of viral RNA revealed some control of post infection viremia. These studies considered week one of infection to be the 1 st week in which viral RNA was detected in plasma (Fig. 4). The one animal infected by the 2 nd series of SIVE660 challenges had a 1000-fold lower level of peak RNA and a 100-fold lower level of viral RNA at 24 weeks post infection than the 8 unvaccinated controls. The SIV251 infected animals had fold lower levels of peak viral RNA (p=0.02, Mann-Whitney test) but no significant reduction at 24 weeks post infection compared to the unvaccinated controls (Fig. 4, C-F). By 24 weeks post infection, RKk11 who had shown a blip associated with seroconversion in the absence of detectable viremia, had become viremic (Fig 3A and 3B, Fig 4D). The serial challenges revealed the DNA/MVA vaccines plus a late MVA boost eliciting remarkably durable protective immunity for SIVE660 (Fig. 1). In addition, the vaccines markedly delayed the acquisition of SIV251 challenges. The poorer protection observed against the SIV251 challenges than the SIVE660 challenges was anticipated because SIV251 is hard to neutralize, whereas SIVE660 is a virus swarm that includes viruses that are neutralization sensitive (7) The data suggest that all six of the extensively challenged animals exhibited local control of SIVE660 infection. This is suggested by transient blips of virus that were associated in three of the six animals with anamnestic mucosal IgA but not IgG responses (Fig. 3). One of the desirable characteristics for a vaccine is the ability to establish a memory response that can undergo a protective anamnestic

5 expansion in response to an infection. Because of the ability of HIV to rapidly establish latent reservoirs and undergo immune escape, it has been proposed that an HIV vaccine will need to be a sterilizing vaccine, or a vaccine that prevents infection in the absence of an anamnestic response. To achieve sterilizing immunity, a vaccine needs to elicit persisting levels of Ab that are sufficient to block an incoming infection. Here, in a series of repeated challenges over a period of 3 1/2 years, we report findings that suggest that a simian DNA/MVA vaccine may not require sterilizing immunity to provide protection, but rather can control rectal challenges by local responses, some of which can be scored as anamnestic IgA responses in rectal secretions. The occurrence of low level blips of virus during serial challenge has also been reported for intravaginal exposures following vaccination with a gp41 virosome vaccine (1). This vaccine elicited Ab in cervical secretions that inhibited trancytosis and showed antibody-dependent cellular cytotoxicity (1). The ability of host immune responses to control local infections has also been suggested for exposed uninfected commercial sex workers and discordant couples (9, 10, 12). Analyses for immune responses in these cohorts have revealed adaptive mechanisms for protection in some individuals (2, 5, 8, 11). In the case of vaccinated people, adaptive responses elicited by the vaccine, both in the presence and absence of anamnestic expansion, could contribute to local control of infection. We suggest that this is happening in macaques for both GM-CSF-adjuvanted and non GM-CSF adjuvanted DNA/MVA SIV vaccines. Acknowledgements. We are indebted to S. Reuland and H. Drake-Perrow for expert administrative support. This work was supported by Integrated Preclinical/Clinical AIDS Vaccine Development program project 5U19 AI (HLR) and the Emory University CFAR P30 AI and by the NCRR (currently supported by the Office of Research Infrastructure Programs)/NIH base grant P51OD to the Yerkes National Primate Research Center.

6 Figure 1. Temporal immunizations, challenges and infection outcomes. A. Immunization and challenge schematic. The schematic presents the timing of vaccinations and challenges and the doses in tissue culture infectious doses 50 (TCID 50 ) of the serial rectal challenges. Age matched controls were added to the study at the time of each serial challenge. The SIVE660 challenge was the SIVsmE660 Hirsch200 stock. The SIV251 stock was the Desrosiers-2006 stock, day 9 harvest provided by DAIDS. B-D. Kaplan- Meier curves for % uninfected for the indicated serial challenge. The numbers of animals in each group are indicated in the graphs. Ch, challenge. Figure 2. Effect of a late boost on temporal immune responses in vaccinated and challenged animals. See Figure 1 and Text for detail on vaccination and challenge regimen. A. Avidity of the elicited Envspecific Ab for the 239 Env concanavilin A-captured from Triton-X-100 disrupted virus like particles produced by transient transfection (6) B. Titer of binding Ab for SIV239 gp140 (Immune Technology Corp) estimated against a standard curve of rhesus IgG (6). C. Specific activity (S.A.) for Env-specific IgA (ng of Env-specific IgA per µg of total IgA) in rectal secretions for SIV251 gp130 (Immune Diagnostics, Woburn, MA (6). D, Magnitudes of responding T cells to Gag and Env determined using intracellular cytokine staining for IFN following stimulation with pools of SIV239 peptides (15-mers overlapping by 11)(4). Vaccination groups are indicated above line graphs and the time in trial above boxplots. DgDgMMM and DDMMM indicate two DNA and three MVA immunizations. Boxplots (minimum, maximum whiskers) are in fuchsia for the DgDgMM and turquoise for the DDMM regimens. Statistics for box plots use a Paired T test and are two-sided. Pre-Ch, point taken 2 to 3 weeks pre-challenge Figure 3. Data consistent with the control of local infections during serial challenges. A. Temporal levels of viral RNA in plasma during the 2 nd through 4 th series of challenges. Levels of viral RNA were tested using a qpcr assay with a sensitivity of 40 and 80 copies per ml for SIVE660 and SIV251 respectively. All blips were confirmed in independent assays. B. Tests for post infection anamnestic

7 serum IgG responses during serial challenges (see Fig. 2). C. Number of blips in different animals during the serial challenges and specific activities of rectal IgA and IgG at the indicted times. The cut-off value for the specific activity of IgA was Fold increases in the specific activity were calculated for week 165 relative to week 137. A 3-fold increase in specific activity is considered significant. Mucosal Ab responses were measured against SIV251 gp130 as previously described (4, 6). Pre-Ch, pre-challenge Figure 4. Temporal levels of post infection viral RNA. Panels A and B present data for the 2 nd challenge with SIVE660 and panels C-F for the 4 th challenge with SIV251. A and C show data for individual unvaccinated animals, B, D and E for individual vaccinated animals and F for the geometric means of data for each group. RNA levels were determined as described in Figure 3. SIV251 viral load is not available for 3 vaccinated animals at week 24 because these animals were euthanized before that time point.

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