JAIDS Journal of Acquired Immune Deficiency Syndromes:
Tonsillar Application of AT-2 SIV Affords Partial Protection Against Rectal Challenge With SIVmac239
Vagenas, Panagiotis PhD*; Williams, Vennansha G BS*; Piatak, Michael Jr PhD†; Bess, Julian W Jr MS†; Lifson, Jeffrey D MD†; Blanchard, James L DVM, PhD‡; Gettie, Agegnehu BS§; Robbiani, Melissa PhD*
From the *Center for Biomedical Research, HIV/AIDS Program, Population Council, New York, NY; †AIDS and Cancer Virus Program, SAIC-Frederick, Inc, National Cancer Institute, Frederick, MD; ‡Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, LA; and §Aaron Diamond AIDS Research Center, Rockefeller University, New York, NY.
Received for publication May 28, 2009; accepted July 20, 2009.
Supported by the National Institutes of Health grant R01 DE016256, the base grant RR00164, and in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01 CO 124000.
Presented at the International Congress of Immunology, August 2007, Rio de Janeiro, Brazil; and at the Modern Mucosal Vaccines, Adjuvants, and Microbicides conference, October 2008, Porto, Portugal.
M.R. was a 2002 Elizabeth Glaser Scientist.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.jaids.com).
Correspondence to: Melissa Robbiani, PhD, Population Council, 1230 York Avenue, New York, NY 10065 (e-mail email@example.com).
Background: Although mucosal responses are important for preventing infections with HIV, the optimal strategies for inducing them remain unclear. To evaluate vaccine strategies targeting the oral mucosal lymphoid tissue inductive sites as an approach to provide immunity at distal sites, we vaccinated healthy macaques via the palatine/lingual tonsils with aldrithiol 2 (AT-2) inactivated Simian immunodeficiency virus (SIV)mac239, combined with CpG-C immunostimulatory oligonucleotide (CpG-C ISS-ODN, C274) as the adjuvant.
Methods: Macaques received 5 doses of C274 or control ODN C661 and AT-2 SIV on the tonsillar tissues every 6 weeks before being challenged rectally with SIVmac239, 8 weeks after the last immunization.
Results: Although no T-cell or B-cell responses were detected in the blood before challenge, antibody (Ab) responses were detected in the rectum. Immunization with AT-2 SIV significantly reduced the frequency of infection compared with nonimmunized controls, irrespective of adjuvant. In the vaccinated animals that became infected, peak viremias were somewhat reduced. SIV-specific responses were detected in the blood once animals became infected with no detectable differences between the differently immunized groups and the controls.
Conclusion: This work provides evidence that vaccine immunogens applied to the oral mucosal associated lymphoid tissues can provide benefit against rectal challenge, a finding with important implications for mucosal vaccination strategies.
A quarter of a century after HIV-1 was shown to be the etiologic agent of AIDS, the pandemic has claimed tens of millions of lives. Despite a global scientific effort, an effective prophylactic vaccine for HIV/AIDS remains elusive. Hopes were dashed again with results from a recent clinical trial not only showing a lack of efficacy in preventing HIV acquisition but also suggesting an increased risk of infection in some vaccinees.1 These results led to recommendations for a renewed emphasis on basic preclinical research and the need to “make best use of animal, particularly monkey, models.”2 The benefits of the SIV/macaque model as the principal animal model for studying HIV are well documented.3
HIV infection occurs mainly via mucosal surfaces, suggesting that induction of mucosal immune responses may be an important property for an effective vaccine. Numerous studies in rodents have provided evidence that oral/nasal administration induces immunity at distal mucosal sites, and systemically. Oral or nasal immunization of mice against Chlamydia or herpes simplex virus (HSV) led to protective immunity in the vagina4-7 and to memory cytotoxic T lymphocyte (CTL) responses.8,9 Mucosal immunization of mice with Tat-containing or Gag-containing vectors led to vaginal protection against challenge with recombinant HIV protein-expressing vectors.10,11 A number of mucosal immunization studies in macaques have also been shown to elicit mucosal and systemic immunity.12-17
Aldrithiol 2 (AT-2)-inactivated SIV and HIV are attractive vaccine immunogens as they contain all of the virion-associated proteins in the absence of an infectious virus.18 Previous work has shown that AT-2 viruses interact authentically with dendritic cells (DCs),19 and mature DCs elicit both CD4+ and CD8+ T-cell responses in vitro.20 Moreover, in therapeutic immunization regimens, injection of autologous mature DCs pulsed with AT-2 SIV or HIV were reported to boost immunity and reduce viral loads in infected macaques21 and in a preliminary clinical study in humans,22 respectively.
CpG-C immunostimulatory oligonucleotides (ISS-ODNs) activate both plasmacytoid dendritic cells (PDCs) and B cells,23,24 to potentially augment innate and adaptive immunity elicited against a vaccine. Similar observations have been made in macaques where CpG-C ISS-ODNs induced PDC activation, interferon alpha (IFNα) and interleukin-12 production, and boosted SIV-specific T-cell responses in vitro25 and stimulating robust B-cell proliferation, survival, and activation.26 Injection of CpG-C ISS-ODNs in macaque lymph nodes also activated both DCs and B cells, demonstrating the ability of CpG-C ISS-ODNs to work in vivo.27 Although CpG ISS-ODNs have been shown to boost macaque immunity,28-30 there is no evidence for the activity of the more broadly acting CpG-C ISS-ODNs in this species.
We examined whether applying a combination of AT-2 SIV and CpG-C ISS-ODNs to the tonsillar mucosa (as a controlled way to model targeting the nasal lymphoid tissues) would protect against rectal challenge with infectious SIV. Although partial protection by tonsillar application of AT-2 SIV was observed, CpG-C ISS-ODNs did not augment this effect suggesting that alternative adjuvant strategies will be needed to optimize the efficacy of mucosally applied AT-2 SIV.
MATERIALS AND METHODS
CpG-C ISS-ODN C274 and the control ODN C661 were provided by Dynavax Technologies (Berkeley, CA). The sequences were: C274 5′-TCGTCGAACGTTCGAGATGAT-3′ and C661 5′-TGCTTGCAAGCTTGCAAGCA-3′. AT-2 SIV (AT-2 SIVmac239 lot numbers: P4001, P4146, P3876, P3778, P3782; AT-2 SIVmac239ΔV1V231 lot number: P3956) and the no virus microvesicle (MV) controls (lot numbers: P3826, P3971), prepared from the same cell line in which the viruses were grown (SUPT1), were provided by the AIDS and Cancer Virus Program (NCI-Frederick, Frederick, MD). AT-2 inactivation of virus was performed as previously described.32 AT-2 SIV was used at 300 ng of p27/mL for all in vitro cultures. MV were normalized to SIV on total protein (300 ng of p27 equivalent/mL). Concanavalin A (ConA; Sigma, St Louis, MO) was used at 1 μg/mL.
Animals and Treatment
Adult male Chinese Rhesus macaques (Macaca mulatta) were housed at the Tulane National Primate Research Center (TNPRC; Covington, LA). All studies were approved by the Animal Care and Use Committee of the TNPRC. The animals' average age at the beginning of the study was 5 years and their average weight was 10 kg. All animals tested negative for simian type D retroviruses, simian T-cell leukemia virus-1, and SIV before use. Animals were anesthetized prior and during all procedures (10 mg ketamine-HCl/kg), in compliance with the regulations detailed under the Animal Welfare Act and in the Guide for the Care and Use of Laboratory Animals.33,34 Animals were immunized a total of 5 times, at 6-week intervals, by application across the lingual and palatine tonsils of 1 mg of CpG-C ISS-ODN C274 or the control ODN C661 mixed with 5 μg of p27 of either AT-2 SIVmac239 or AT-2 SIVmac239ΔV1V231 in a volume of 100 μL. The treatment groups included animals immunized with the following: C274 and wild-type AT-2 SIVmac239 (C274/wt), C274 and AT-2 SIVmac239ΔV1V2 (C274/V1V2), C661 and wild-type AT-2 SIVmac239 (C661/wt), C661 and AT-2 SIVmac239ΔV1V2 (C661/V1V2), or nothing (nonvaccinated controls). Table 1 lists all study animals. Eight weeks after the final immunization, animals were challenged rectally with 103 50% tissue culture infectious dose (TCID50) of SIVmac239 (TNPRC stock virus propagated in staphylococcal enterotoxin B (SEB)-stimulated rhesus peripheral blood mononuclear cells (PBMCs); “SIVmac239 RhPBMC 7/29/94”). Once the follow-up period was completed, 5 months after the initial challenge, uninfected animals were reimmunized once more, rechallenged 8 weeks later, and followed up for 6 months (as indicated). Immune responses were followed by collecting EDTA-anticoagulated blood and mucosal (oral and rectal) fluids throughout the immunization regimen and for up to 6 months after SIV challenge. Mucosal fluids were collected by insertion of a foam pad (approximately size 1 × 0.5 cm) in the mucosal cavity for 5 minutes, after which the swab was placed into a tube containing 1 mL phosphate buffer saline (PBS)/1% fetal calf serum (FCS)/penicillin-streptomycin (Cellgro/Mediatech, VA). Blood, fluids, and tissue samples were transported to the laboratory by overnight courier service. Blood was processed as described below, and the mucosal fluids were spun at 805g for 10 minutes, collecting the supernatant and storing at −80°C until analysis. Upon study termination, animals were sacrificed and standard full necropsy for SIV-infected animals was performed. To further assess the in vivo activity of mucosally applied CpG-C ISS-ODNs, 1 mg of C274 or C661 (50 μL of 20 mg/mL stock) were applied to the tonsils of healthy infected or uninfected macaques, and tonsillar pinch biopsies were collected 24 hours later. Cellular activation was monitored by flow cytometry.
Macaque PBMCs were isolated using Ficoll-Hypaque density centrifugation (GE Healthcare, Uppsala, Sweden). Cells were cultured in complete RPMI 1640 (Cellgro, Springfield, NJ) containing 2 mM L-glutamine (GIBCO Life Technologies, Grand Island, NY) 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; GIBCO Life technologies), 50 μM 2-mercaptoethanol (Sigma), penicillin (100 U/mL)/streptomycin (100 μg/mL) (GIBCO Life Technologies), and 1% heparinized human plasma (Innovative Research, Southfield, MI).
Tonsillar biopsies were placed in RPMI [supplemented as above, but with 10% heat-inactivated fetal bovine serum (Mediatech, Manassas, VA) instead of human plasma] containing 200 μg/mL gentamycin (GIBCO) for 1 hour at 4°C. The tissue was washed by spinning at 244g for 10 minutes and resuspending in medium (RPMI-10% FBS) containing 400 U/mL Collagenase D (Roche, Indianapolis, IN) and 10 μg/mL DNAse I (Roche) in a tissue culture dish. The tissue was broken up using a forceps and a scalpel and incubated at 37°C for 1 hour. It was then filtered using a 70 μm nylon filter (BD Falcon), and the suspension was spun at 340g for 10 minutes. Cells were then resuspended in RPMI (1% human plasma) and counted.
Four-color flow cytometry was used to characterize leukocyte subsets from macaque blood and tissue. DCs were identified as Lin−HLA-DR+ populations using fluorescein isothiocyanate (FITC)-conjugated antilineage marker Abs (CD3, CD8, CD11b, clones SP34, SK1, and F6.2); CD14, clone M5E2 (BD Biosciences); CD20, clone L27 (BD Biosciences) and antigen presenting cell (APC)-conjugated anti-HLA-DR (clone G46-6; BD Biosciences, San Jose, CA). PDCs and myeloid dendritic cell (MDC)-containing fractions were identified as CD123+ and CD123− subsets within the Lin−HLA-DR+ cells, respectively, using PE-conjugated anti-CD123 (clone TU27; BD Biosciences). DC activation status was examined using Cy-conjugated anti-CD86 (clone 2331 FUN1; BD Biosciences) and anti-CD80 (clone L307.4; BD Biosciences). B cells were identified using FITC-conjugated anti-CD20 and their activation status was monitored using PE-conjugated anti-CD86 (clone IT2.2; BD Biosciences), anti-CD80, and anti-CD40 (clone 5C3; BD Biosciences). T-cell subsets were identified using APC-conjugated anti-CD28 (clone CD28.2, Biolegend) and PE-conjugated CD95 (clone DX2; BD Biosciences).
The appropriate isotype immunoglobulin (Ig) controls were included in all experiments and typically gave mean fluorescence intensity (MFIs) of <1 log. Samples were acquired on a FACSCalibur (BD) and analyzed using FlowJo software (Tree Star, OR).
Numbers of IFN-γ spot-forming cells (SFCs) in peripheral blood responding to wild-type AT-2 SIVmac239, to AT-2 SIVmac239ΔV1V2 mutant or Gag/Env peptide pools (National Institutes of Health AIDS Research and Reference Reagent Program) were measured by ELISPOT.25 Each peptide pool was made by mixing 10 consecutive peptides (resuspended in 100 μL dimethyl sulfoxide [DMSO]) at 2 μg/mL for each peptide (0.2% DMSO final concentration). Twenty-two peptide pools were prepared for Env and 13 for Gag, covering the entire span of each protein. ConA was used at 1 μg/mL as a positive control. Medium, MV, and 0.2% DMSO controls were included for the respective stimuli.
Viral Load and SIV Ab Detection
Plasma samples were collected from all animals at all time points of the study by centrifuging whole blood at 805g for 10 minutes, collecting the clear supernatant, centrifuging again, and aliquots of the supernatants were stored at −80°C. SIV RNA was determined by quantitative reverse transcriptase-polymerase chain reaction,35 and SIV-specific Abs were measured by enzyme-linked immunosorbent assay (ELISA).36
Neutralizing Ab activity against SIV was measured in monkey plasma samples using minor adaptations to published protocols.37,38 Plasma, from weeks 8-10 postinfection, was heat inactivated by incubating at 56°C for 1 hour and then clarified by centrifugation at 956g. Samples were then diluted 5-fold twice in flat-bottom 96-well plates (BD Falcon, Franklin Lakes, NJ). Fifty TCID50 of SIVmac239 or SIVmac251 were added to each plasma-containing well and incubated for 1 hour at 37°C. 3 × 105 174xCEM cells (National Institutes of Health AIDS Research and Reference Reagent Program) were added per well, and the plates were incubated for 2 weeks at 37°C. Fifty microliters of culture medium were exchanged for fresh medium after 7 days. No more plasma was added during this period. Samples were run in duplicate. The plates were monitored for cytotoxicity every 3 days, and cell-free supernatants were collected at day 14 of culture for p27 ELISA (ZeptoMetrix Corporation, NY). Pooled heat-inactivated plasma from SIVmac239Δnef/wild-type-infected animals (healthy, long-term infected) was used as a positive control. Negative controls included plasma from uninfected monkeys, and wells with no plasma added.
SIV-specific IgA was measured in rectal fluids collected at the beginning of the study, and at the last time point before challenge (week 26) or after challenge (week 36), by ELISA as previously described,17 with minor modifications. Briefly, 96-well plates (Costar, NY) were coated overnight with lysed SIVmac239 (lot P4145) at 1 μg/mL. Plates were blocked with 0.25% gelatin in PBS, the samples were added (100 μL per well) and incubated for 2 hours at 37°C. Peroxidase-conjugated goat antimonkey IgA (Alpha Diagnostic International, San Antonio, TX) was used as the secondary Ab at 1:10,000 and incubated for 2 hours at 37°C. Tetramethylbenzidine (TMB) peroxidase substrate solution (KPL, Gaithersburg, MD) was then added (100 μL per well) and incubated for 30 minutes at room temperature. The reaction was stopped with 1M HCl (50 μL per well), and the absorbance was read at 450/650 nm. Pooled plasma from SIVmac239Δnef/wild-type-infected animals (healthy, long-term infected) was used as a positive control. Plasma from uninfected monkeys was used as a negative control.
Viral load data were analyzed for statistical significance using the Mann-Whitney test. Frequency of infection data were analyzed using the Fisher exact test. The P values <0.05 were taken as statistically significant.
Tonsillar C274/AT-2 SIV Vaccination Partially Protected Against SIV Challenge
Knowing that C274 activates macaque DC and B cells and augments SIV-specific T-cell responses,25-27 we set out to determine if combining C274 and AT-2 SIV would serve as a potent vaccine when applied to the oral mucosal associated lymphoid tissues (MALT) of macaques. This allowed us to control that the vaccine was directly applied to the oral MALT (and not swallowed) to provide a model for future strategies that would target nasal MALT. In addition to comparing C274 as the adjuvant, we compared 2 vaccine antigens, wild-type AT-2 SIVmac239 and AT-2 SIVmac239ΔV1V2,31 a mutant of the virus that lacks the hypervariable loops V1 and V2 from the viral envelope. We hypothesized that the deletion of V1 and V2 in the mutant virus might reveal neutralization sensitive epitopes that would be presented in this AT-2-treated vaccine, thereby inducing more potent neutralizing Ab responses, although inducing a similar cellular response to the wild-type virus.
SIV-naive Chinese rhesus macaques were vaccinated 5 times by applying AT-2 SIV with the indicated ODNs to the tonsillar tissues every 6 weeks (Fig. 1). Eight weeks after the final immunization, the animals were challenged rectally with pathogenic SIVmac239. They were followed up for a period of 6 months. Vaccinated animals that remained uninfected after 5 months of follow-up were then reimmunized once more and rechallenged 8 weeks later and followed for an additional 6 months to see if they continued to resist infection (Table 1). When comparing all challenges and infection outcomes, the vaccinated animal groups exhibited a significantly lower frequency of infection (average of 53%) compared with the nonvaccinated control group (83%), independent of the presence of C274 (Fig. 2, Table 1; P < 0.03 when comparing all challenges for each group vs control).
Examination of the plasma viral loads (Fig. 3) revealed that all infected control animals, 6 of 6 infected C274/wt-vaccinated animals and 2 of 4 infected C661/wt-vaccinated animals exhibited peak viremia 2 weeks post challenge. Peak viremias were delayed by 1-2 weeks in 4 of 6 infected C274/V1V2 and 1 of 2 infected C661/V1V2-vaccinated animals. Closer analysis of acute viremia revealed that wild-type-vaccinated animals showed significantly lower viremia than controls (independent of C274) during the first week of infection (Fig. 4, upper panel) but no difference in subsequent weeks. ΔV1V2 vaccinees also had significantly lower viremia than controls during the first 2 weeks of infection, with the exception of C661 vaccinees in the first week (Fig. 4, lower panel). Setpoint viremias (weeks 8 and 10 postinfection) and viremias at time of necropsy (4-11 months post infection) yielded no significant differences between any of the groups.
Blood CD4+ T-cell counts were measured over the length of the study, but there were no differences in the CD4 decline between the groups (Table 1). Full simian AIDS necropsies were performed when possible, and the pathology results are summarized in an online table (see Supplemental Digital Content 2, http://links.lww.com/QAI/A25). No dramatic pathology or striking differences were observed between groups. All animals, with one exception, survived the 6-month follow-up period. The animal (GJ66) that died during this period was in the C661/wt virus vaccination group and was sacrificed at 15 weeks postinfection due to excessive weight loss, a sign of simian AIDS. The CD4 decline observed in this animal (403 cells/μL blood at necropsy) was similar in magnitude to other animals.
C274 Did Not Alter Innate and Adaptive Immune Responses in vivo
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.
1. Steinbrook R. One step forward, two steps back-will there ever be an AIDS vaccine? N Engl J Med. 2007;357:2653-2655.
2. Walker BD, Burton DR. Toward an AIDS vaccine. Science. 2008;320:760-764.
3. Desrosiers RC. The simian immunodeficiency viruses. Ann Rev Immunol. 1990;8:557-578.
4. Cui ZD, Tristram D, LaScolea LJ, et al. Induction of antibody response to Chlamydia trachomatis in the genital tract by oral immunization. Infect Immun. 1991;59:1465-1469.
5. Pal S, Peterson EM, de la Maza LM. Intranasal immunization induces long-term protection in mice against a Chlamydia trachomatis genital challenge. Infect Immun. 1996;64:5341-5348.
6. Gallichan WS, Johnson DC, Graham FL, et al. Mucosal immunity and protection after intranasal immunization with recombinant adenovirus expressing herpes simplex virus glycoprotein B. J Infect Dis. 1993;168:622-629.
7. Milligan GN, Dudley-McClain KL, Chu CF, et al. Efficacy of genital T cell responses to herpes simplex virus type 2 resulting from immunization of the nasal mucosa. Virology. 2004;318:507-515.
8. Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med. 1996;184:1879-1890.
9. Gallichan WS, Rosenthal KL. Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization. J Infect Dis. 1998;177:1155-1161.
10. Dumais N, Patrick A, Moss RB, et al. Mucosal immunization with inactivated human immunodeficiency virus plus CpG oligodeoxynucleotides induces genital immune responses and protection against intravaginal challenge. J Infect Dis. 2002;186:1098-1105.
11. Adalid-Peralta L, Godot V, Colin C, et al. Stimulation of the primary anti-HIV antibody response by IFN-alpha in patients with acute HIV-1 infection. J Leukoc Biol. 2008;83:1060-1067.
12. Vajdy M, Singh M, Kazzaz J, et al. Mucosal and systemic anti-HIV responses in rhesus macaques following combinations of intranasal and parenteral immunizations. AIDS Res Hum Retroviruses. 2004;20:1269-1281.
13. Wang SW, Bertley FM, Kozlowski PA, et al. An SHIV DNA/MVA rectal vaccination in macaques provides systemic and mucosal virus-specific responses and protection against AIDS. AIDS Res Hum Retroviruses. 2004;20:846-859.
14. Pahar B, Cantu MA, Zhao W, et al. Single epitope mucosal vaccine delivered via immuno-stimulating complexes induces low level of immunity against simian-HIV. Vaccine. 2006;24:6839-6849.
15. Bogers WM, Davis D, Baak I, et al. Systemic neutralizing antibodies induced by long interval mucosally primed systemically boosted immunization correlate with protection from mucosal SHIV challenge. Virology. 2008;382:217-225.
16. Schulte R, Suh YS, Sauermann U, et al. Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques. Virology. 2009;383:300-309.
17. Hidajat R, Xiao P, Zhou Q, et al. Correlation of vaccine-elicited systemic and mucosal non-neutralizing antibody activities with reduced acute viremia following intrarectal SIVmac251 challenge of rhesus macaques. J Virol. 2009;83:791-801.
18. Lifson JD, Piatak M Jr, Rossio JL, et al. Whole inactivated SIV virion vaccines with functional envelope glycoproteins: Safety, immunogenicity, and activity against intrarectal challenge. J Med Primatol. 2002;31:205-216.
19. Frank I, Piatak MJ, Stoessel H, et al. Infectious and whole inactivated simian immunodeficiency viruses interact similarly with primate dendritic cells (DCs): differential intracellular fate of virions in mature and immature DCs. J Virol. 2002;76:2936-2951.
20. Frank I, Santos JJ, Mehlhop E, et al. Presentation of exogenous whole inactivated simian immunodeficiency virus by mature dendritic cells induces CD4+ and CD8+ T cell responses. J AIDS. 2003;34:7-19.
21. Lu W, Wu X, Lu Y, et al. Therapeutic dendritic-cell vaccine for simian AIDS. Nat Med. 2003;9:27-32.
22. Lu W, Arraes LC, Ferreira WT, et al. Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat Med. 2004;10:1359-1365.
23. Vollmer J, Weeratna R, Payette P, et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 2004;34:251-262.
24. Marshall JD, Fearon K, Abbate C, et al. Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions. J Leuk Biol. 2003;73:781-792.
25. Teleshova N, Kenney J, Jones J, et al. CpG-C immunostimulatory oligodeoxyribonucleotide activation of plasmacytoid dendritic cells in rhesus macaques to augment the activation of IFN-gamma-secreting simian immunodeficiency virus-specific T cells. J Immunol. 2004;173:1647-1657.
26. Teleshova N, Kenney J, Van Nest G, et al. CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques. J Leuk Biol. 2006;79:257-267.
27. Teleshova N, Kenney J, Van Nest G, et al. Local and systemic effects of intranodally injected CpG-C ISS-ODNs in macaques. J Immunol. 2006;177:8531-8541.
28. Verthelyi D, Kenney KT, Seder RA, et al. CpG oligodeoxynucleotides as vaccine adjuvants in primates. J Immunol. 2002;168:1659-1663.
29. Hartmann G, Weeratna RD, Ballas ZK, et al. Delineation of a CpG phosphorothionate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J Immunol. 2000;164:1617-1624.
30. Cafaro A, Titti F, Fracasso C, et al. Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P). Vaccine. 2001;19:2862-2877.
31. Johnson WE, Lifson JD, Lang SM, et al. Importance of B cell responses for immunological control of variant strains of simian immunodeficiency virus. J Virol. 2003;77:375-381.
32. Rossio JL, Esser MT, Suryanarayana K, et al. Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins. J. Virol. 1998;72:7992-8001.
33. Animal Welfare Act and Regulation. Code of Federal Regulations. Chapter 1. In: Animals and Animal Products.
34. Guide for the Care and Use of Laboratory Animals. Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources UdoHaHS; 1985:1-83.
35. Cline AN, Bess JW, Piatak M Jr, et al. Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS. J Med Primatol. 2005;34:303-312.
36. Smith SM, Holland B, Russo C, et al. Retrospective analysis of viral load and SIV antibody responses in rhesus macaques infected with pathogenic SIV: predictive value for disease progression. AIDS Res Hum Retroviruses. 1999;15:1691-1701.
37. Horton H, Vogel TU, Carter DK, et al. Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239. J. Virol. 2002;76:7187-7202.
38. Cole KS, Rowles JL, Jagerski BA, et al. Evolution of envelope-specific antibody responses in monkeys experimentally infected or immunized with simian immunodeficiency and its association with the development of protective immunity. J Virol. 1997;71:5069-5079.
39. Stahl-Hennig C, Steinman RM, Tenner-Racz K, et al. Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus. Science. 1999;285:1261-1265.
40. Baba TW, Koch J, Mittler ES, et al. Mucosal infection of neonatal rhesus monkeys with cell-free SIV. AIDS Res Hum Retroviruses. 1994;10:351-357.
41. Cao J, Sullivan N, Desjardin E, et al. Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein. J Virol. 1997;71:9808-9812.
42. Johnson WE, Morgan J, Reitter J, et al. A replication-competent, neutralization-sensitive variant of simian immunodeficiency virus lacking 100 amino acids of envelope. J Virol. 2002;76:2075-2086.
43. Stamatatos L, Cheng-Mayer C. An envelope modification that renders a primary, neutralization-resistant clade B human immunodeficiency virus type 1 isolate highly susceptible to neutralization by sera from other clades. J Virol. 1998;72:7840-7845.
44. Zhou Q, Hidajat R, Peng B, et al. Comparative evaluation of oral and intranasal priming with replication-competent adenovirus 5 host range mutant (Ad5hr)-simian immunodeficiency virus (SIV) recombinant vaccines on immunogenicity and protective efficacy against SIV(mac251). Vaccine. 2007;25:8021-8035.
45. Hogrefe WR. Biomarkers and assessment of vaccine responses. Biomarkers. 2005;10(Suppl 1):S50-S57.
46. Allen TM, Mortara L, Mothe BR, et al. Tat-vaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication. J Virol. 2002;76:4108-4112.
47. Allen TM, Jing P, Calore B, et al. Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection. J Virol. 2002;76:10507-10511.
48. Ahmad S, Lohman B, Marthas M, et al. Reduced virus load in rhesus macaques immunized with recombinant gp160 and challenged with simian immunodeficiency virus. AIDS Res Hum Retroviruses. 1994;10:195-204.
49. Mori K, Sugimoto C, Ohgimoto S, et al. Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model. J Virol. 2005;79:10386-10396.
50. Casimiro DR, Wang F, Schleif WA, et al. Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag. J Virol. 2005;79:15547-15555.
51. Kawada M, Tsukamoto T, Yamamoto H, et al. Gag-specific cytotoxic T-lymphocyte-based control of primary simian immunodeficiency virus replication in a vaccine trial. J Virol. 2008;82:10199-10206.
52. John M, Moore CB, James IR, et al. Interactive selective pressures of HLA-restricted immune responses and antiretroviral drugs on HIV-1. Antivir Ther. 2005;10:551-555.
53. Pore N, Gupta AK, Cerniglia GJ, et al. Nelfinavir down-regulates hypoxia-inducible factor 1alpha and VEGF expression and increases tumor oxygenation: implications for radiotherapy. Cancer Res. 2006;66:9252-9259.
54. Turville SG, Aravantinou M, Miller T, et al. Efficacy of Carraguard®-based microbicides in vivo despite variable in vitro activity. PLos ONE. 2008;3(9):e3162.
55. Moseman EA, Liang X, Dawson AJ, et al. Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J Immunol. 2004;173:4433-4442.
56. Ito T, Yang M, Wang YH, et al. Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand. J Exp Med. 2007;204:105-115.
57. Manches O, Munn D, Fallahi A, et al. HIV-activated human plasmacytoid DCs induce Tregs through an indoleamine 2,3-dioxygenase-dependent mechanism. J Clin Invest. 2008;118:3431-3439.
58. Wang Y, Blozis SA, Lederman M, et al. Enhanced antibody responses elicited by a CpG adjuvant do not improve the protective effect of an aldrithiol-2-inactivated simian immunodeficiency virus therapeutic AIDS vaccine. Clin Vaccine Immunol. 2009;16:499-505.
59. Manrique M, Micewicz E, Kozlowski PA, et al. DNA-MVA vaccine protection after X4 SHIV challenge in macaques correlates with day-of-challenge antiviral CD4+ cell-mediated immunity levels and postchallenge preservation of CD4+ T cell memory. AIDS Res Hum Retroviruses. 2008;24:505-519.
60. Martinon F, Brochard P, Ripaux M, et al. Improved protection against simian immunodeficiency virus mucosal challenge in macaques primed with a DNA vaccine and boosted with the recombinant modified vaccinia virus Ankara and recombinant Semliki Forest virus. Vaccine. 2008;26:532-545.
61. Kwissa M, Amara RR, Robinson HL, et al. Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus. J Exp Med. 2007;204:2733-2746.
62. Demberg T, Boyer JD, Malkevich N, et al. Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge. J Virol. 2008;82:10911-10921.
63. Liu J, O'Brien KL, Lynch DM, et al. Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature. 2009;457:87-91.
64. Daniel MD, Kirchhoff F, Czajak SC, et al. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science. 1992;258:1938-1941.
65. Cranage MP, Whatmore AM, Sharpe SA, et al. Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa. Virology. 1997;229:143-154.
66. Mansfield K, Lang SM, Gauduin MC, et al. Vaccine protection by live, attenuated simian immunodeficiency virus in the absence of high-titer antibody responses and high-frequency cellular immune responses measurable in the periphery. J Virol. 2008;82:4135-4148.
67. Spear GT, Takefman DM, Sullivan BL, et al. Anti-cellular antibodies in sera from vaccinated macaques can induce complement-mediated virolysis of human immunodeficiency virus and simian immunodeficiency virus. Virology. 1993;195:475-480.
68. Mothe BR, Weinfurter J, Wang C, et al. Expression of the major histocompatibility complex class I molecule Mamu-A*01 is associated with control of simian immunodeficiency virus SIVmac239 replication. J Virol. 2003;77:2736-2740.
69. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol. 2004;22:745-763.
70. Groot F, van Capel TM, Schuitemaker J, et al. Differential susceptibility of naive, central memory and effector memory T cells to dendritic cell-mediated HIV-1 transmission. Retrovirology. 2006;3:52.
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