Paralleling the measurement of viral parameters, adaptive immune responses were monitored over time by measuring the presence of SIV-specific T-cell and B-cell responses in blood and mucosal fluids. No SIV-specific immune responses were detected in peripheral blood after tonsillar immunization, as measured by numbers of PBMCs producing IFNγ in response to stimulation with AT-2 SIV (ELISPOT) or the presence of SIV-specific Abs in plasma. However, after challenge, comparable SIV-specific T-cell and B-cell responses in blood were detected in most infected animals (Table 1). Both AT-2 SIV wild type and AT-2 SIV ΔV1V2 were used as stimuli in vitro and elicited comparable T-cell responses in all animals (data not shown), indicating that there were limited T-cell responses against the deleted V1V2 region. In vitro Ab neutralization activity against pathogenic SIVmac239 and SIVmac251 was also measured in plasma from the infected animals in each group. As reported by others,37,38 limited neutralization of SIVmac239 was observed, but SIVmac251 was neutralized more effectively (Fig. 5). There seemed to be no difference between any of the groups.
Mucosal SIV-specific IgA was measured in the rectal fluids of animals at the beginning of the study, as a baseline and again before challenge, to determine whether there were vaccine-induced adaptive responses in the mucosa. Five of 7 animals in the C274/wt group, 3 of 4 animals in the C661/wt group, 5 of 6 animals in the C274/V1V2 group, and 3 of 3 animals in the C661/V1V2 group exhibited positive vaccine-induced IgA responses, although the actual titers were low in all cases. For the animals with a positive postvaccination rectal SIV-specific IgA response, the average fold increases, compared with baseline, ranged from 2.2-fold to 3.9-fold (Fig. 6), and there was no significant difference between the differently immunized groups. IgA levels were also measured at 4 weeks postchallenge. The average fold increases ranged from 2.4 to 4 (Fig. 6) and were similar to those seen postvaccination, suggesting that mucosal IgA is primarily a vaccine-induced response. However, a 2.7-fold increase in mucosal IgA was also observed for control animals, suggesting that infection also induces this type of humoral response in naive animals.
To further characterize the SIV-specific immune responses, IFN γ responses to Env and Gag peptide pools were measured, and the percentages of effector vs central memory T-cell subsets were determined at the time of necropsy in some animals (5-10 months postinfection). At this late time point, the average IFNγ response to AT-2 SIV was only 22 SFC/2 × 105 cells. Not surprisingly, minimal Env or Gag peptide-specific responses were seen in all animals tested (1-20 SFC/2 × 105 cells), with no preferential responses to any peptide pool being detected (data not shown). Similarly, there were no significant differences in the numbers of effector memory (CD28−CD95+), central memory (CD28+CD95+), or naive (CD28+CD95−) CD4+ T-cell subsets in the blood or lymphoid tissues of the differently immunized animals (see Figure A, Supplemental Digital Content 1, http://links.lww.com/QAI/A23), no nonvaccinated controls were tested in this experiment. In blood and lymphoid tissues, the percentage of central memory T cells was higher than that of effector memory cells. This was more pronounced in lymphoid tissues, and blood contained a bigger percentage of naive CD28+CD95− T cells. Notably, the CD28−CD95+ effector memory subset (and CD28−CD95−-naive cells) was detected in the ileum, and these levels were comparable to those seen at other sites.
To investigate the lack of significant C274 enhancement of the protective effect of AT-2 SIV in the vaccine observed at the virologic and immunologic levels, we applied C274 or C661 in vivo on the tonsils and monitored local cellular activation. DCs (Lin-HLA-DR+) and Lin+ cells (containing B cells) within the suspensions isolated from pinch biopsies taken 24 hours after ODN application were then monitored for CD80 and CD86 expression (see Figure B, Supplemental Digital Content 1, http://links.lww.com/QAI/A23). CD86 and CD80 expression remained unchanged in both PDC (CD123+) and MDC (CD123-) subsets after application of C274 to the tonsils. A small increase in CD86 expression was observed in the Lin+HLA-DR+ B cell-containing fraction after C274 application (not significant, P = 0.5), whereas CD80 expression remained unchanged. This was not due to the timing because cells from the tonsils of animals receiving C274 vs C661 48 hours before biopsy (n = 3 each) similarly showed no change in DC or B-cell CD80/CD86 expression (data not shown). Thus, the limited activity of C274 in stimulating local DC/B-cell activation after tonsillar application might contribute to its suboptimal ability to boost immunity and the protective effects of AT-2 SIV.
This study aimed to test the efficacy of a CpG-C ISS-ODN/AT-2 SIV-based vaccine applied to oral MALT in preventing rectal SIV infection. Earlier studies have described the effectiveness of oral/nasal vaccines in inducing responses at distal mucosal sites.4,5,10,12 We used the accessible palatine/lingual tonsils to model pharyngeal tonsils likely targeted by nasal vaccines for this proof of concept approach against SIV. CpG-C ISS-ODNs are effective activators of B cells and DCs23,24 and boost SIV-specific T-cell responses in vitro.25 AT-2-inactivated virus contains all of the virion proteins but is noninfectious.18 Mature DCs presenting AT-2 SIV/HIV stimulate CD4+ and CD8+ T cells in vitro,20 and AT-2 virus-loaded mature DCs showed promise as a therapeutic vaccine.22 Because animals become infected after tonsillar application of infectious SIV39,40 and AT-2 SIV interacts authentically with target cells,19 we hypothesized that AT-2 SIV applied to the tonsils would cross the epithelial barriers, enter the underlying MALT, and induce virus-specific immunity. Two types of AT-2-inactivated SIVmac239 virus were used here: the wild type and the V1V2 mutant, where the hypervariable loops V1 and V2 of the viral envelope protein have been deleted. The deletion in the latter reveals neutralization sensitive epitopes.41-43 We postulated that the exposed neutralization face might be presented within the AT-2-treated form to induce a superior Ab response compared with the wild-type form, although also inducing similar T-cell responses, thereby leading to greater control of infection than the wild-type AT-2 SIV.
Targeting the MALT by vaccinating across the palatine/lingual tonsils provided a controlled way in which we could test the immunogenicity of CpG-C ISS-ODN/AT-2 SIV as a mucosal vaccine. Although SIV-specific T-cell and B-cell responses were not detected in the blood after immunization, application of AT-2 SIV to the tonsils protected 53% of the animals from infection by subsequent rectal challenge with pathogenic SIVmac239. It has been previously reported that no nasal vaccine-induced PBMC IFNγ responses were measured before significant post rectal challenge protection.44 Also, the value of the IFNγ ELISPOT as a sole measure of immune activation has been debated in the past.45
Given the rectal route of challenge used here, SIV-specific IgA were measured in the rectal fluids, before challenge. Low-level vaccine-induced SIV-specific Abs were identified in most vaccinees, even though no such Abs were seen in the plasma before infection. Rectal SIV-specific IgA were increased from 2.2-fold to 3.9-fold in all groups, compared with baseline levels in line with previous publications.17 As with most other data presented here, no differences were observed between vaccination groups, suggesting that the vaccine effect observed is due to the AT-2 SIV, irrespective of adjuvant or form of the virus. The presence of these Abs in the prechallenge mucosal fluids of the vaccinated animals may contribute to the vaccine protective effect observed, although no direct correlation between Ab levels and infection was determined. SIV-specific IgA responses were not boosted when vaccinated animals became infected, although comparable responses were detected in nonvaccinated controls after infection.
SIVmac239 is commonly used in preclinical vaccine trials but has so far been shown to be very difficult to protect against as a homologous or heterologous intravenous or rectal challenge.37,46-49 The magnitude of the viral inocula for mucosal challenge with our SIVmac239 stock was comparable to what was used previously46,50,51 and has also been historically very effective in our laboratory in infecting naive animals via the rectal route (frequency of infection >90%; 29 of 32 challenged monkeys infected). Notably, we observed that 53% of the AT-2 SIV-vaccinated animals were protected from homologous mucosal challenge with SIVmac239. AT-2 SIV-vaccinated animals had lower rates of infection than controls, irrespective of C274 or C661 as an adjuvant and irrespective of whether AT-2 wild-type or V1V2 virus was used as the vaccine. The discordance between previously published in vitro activities of C27425,26 and the apparent lack of vaccine enhancement by C274 observed herein can be due to a number of reasons. First, differences between in vitro and in vivo observations are not uncommon52-54 and can be due to numerous factors, for example, the active component perhaps cannot efficiently cross the epithelial cell layer to activate the underlying leukocytes. Another possibility is that CpGs, via their effect on PDCs, induce regulatory T cells (Tregs) that suppress further immune activation.55,56 It has been recently shown that HIV-stimulated human PDCs can also induce Treg generation 57. Even though Tregs were not measured here, the lack of differences in the immune responses measured suggests that Treg activation by C274 is unlikely to have played a significant role. A recent study using CpG-B and AT-2 SIV as a therapeutic vaccine in SIV-infected macaques also showed a lack of enhancement of the AT-2 SIV effect by the CpG.58
AT-2 SIVmac239 (wild type or V1V2 mutant) applied on the tonsils confers a significant protective effect against pathogenic challenge at the distal rectal mucosa. Many previous macaque vaccine studies, using varied vaccination techniques, such as DNA prime/modified vaccinia ankara (MVA) boost or adenoviral prime/boost, show development of humoral and cellular postimmunization immune responses in the blood, which have not been observed here. In these studies though all animals became infected upon pathogenic SIV challenge,51,59-63 on the other hand, vaccine studies with live attenuated viruses64-66 or other replication-competent viruses44 seem to show significant protection against pathogenic SIV challenge, but safety concerns may limit translating such research to humans. Not only was the AT-2 SIV vaccine partially effective, it is safer than replicating vaccines. The possibility that cellular components in the vaccine virus preparation67 might have an effect was not addressed by this study, nevertheless, the presence of SIV-specific responses (especially the rectal IgA responses postvaccination) suggest that the vaccine effect is due to SIV antigens. Also, the genetic background of the animals was not examined. However, it seems unlikely that the random assignment of animals to vaccine vs control groups would have divided them into groups with different genetic susceptibilities.68
Although there was no difference in the frequency of infection between the vaccinated groups, most of the V1V2-vaccinated animals exhibited delayed peak viremia. Significant differences were also observed between the average viral loads of vaccinees, compared with controls, during the first 2 weeks of infection, suggesting a slight delay in viral replication, which is then overcome by the third week of infection. We had hypothesized that the V1V2 mutant might induce more neutralizing Abs,43 which could contribute to improved control of infection, but there was no difference in the neutralizing Ab activity (in plasma) nor the rectal IgA Ab responses detected between the different groups.
Despite the reduced infection frequency in the AT-2 SIV-vaccinated animals, no considerable differences were observed between groups once the animals were infected, as measured by plasma viremia, CD4 counts, numbers of IFNγ-producing cells, and disease progression. There were also no differences in the distribution of effector and central memory CD4+ T cells between vaccinated groups, with central memory cells dominating in the blood and lymphoid tissues and effector memory cells predominating in the gut, as expected.69,70
This study suggests that tonsillar immunization with a nonreplicating immunogen can help protect against rectal challenge with a highly pathogenic SIV, although we could not correlate SIV-specific immune responses with protection. In addition to reducing the frequency of infection, AT-2 SIVΔV1V2 seemed to better limit the initial amplification of infection in some animals. Although C274 seemed to have no boosting effect on the AT-2 SIV vaccination under this regimen, future studies using this and/or other toll-like receptor ligands to augment oral/nasal vaccines represent an exciting strategy to tackle HIV.
174xCEM cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, courtesy of Peter Cresswell. SIV Env and Gag peptide pools were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. We thank William Bohn, Jeremy Miller, Terra Schaden-Ireland, Rodman Smith, Robert Imming, and Elena Chertova for producing, inactivating, purifying, and characterizing AT-2 SIV and MV preparations. We thank Jason Marshall and Gary Van Nest from Dynavax Technologies for the ODNs. We would like to acknowledge the Population Council Cell Core for flow cytometry assistance and the veterinary staff at the TNPRC for their continued support. We thank Irving Sivin (Population Council) for his assistance with statistical analyses and R. Paul Johnson (New England Primate Research Center, Harvard University) for advice on the Env/Gag peptide pools. We thank members of our laboratory for their assistance in editing the article and continued help during the course of this study.
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