Efficacy studies in humans and in experimental simian immunodeficiency virus (SIV) infection of monkeys support the idea that an effective HIV vaccine should induce strong, lasting, and protective immunity.1,2 But human efficacy trials require large numbers of volunteers drawn mainly from high-risk groups and rely on accidental exposure to allow for stringent evaluation of the outcome.3 Consequently, preclinical studies using the SIV/macaque model are requisites to candidate vaccine testing.4,5 We earlier reported strong vaccine-induced SIV-specific interferon gamma (IFNγ) responses that were associated with reduced viremia in macaques.6 Others observed protection to be related to improved cytotoxic T-lymphocyte (CTL) and antibody responses.7,8 Evidently, polyfunctional T cells producing multiple cytokines are good indicators for vaccine efficacy and susceptibility to infection.9-11 However, the recent failure of the Ad5-HIV-based phase 3 “Step” vaccine trial,12 exemplifies the gap between strong immunological response found in the preceding efficacy and safety evaluations13-15 and actual protection. Hence, new tests are needed which can better predict the effects of the vaccines on virus replication and disease progression before actual preclinical monkey challenge experiments or large clinical efficacy trials in humans.
Antiviral activity is not always associated with cytokine production by T cells.16 Throughout the course of infection, antiviral activities can be observed without evident cytotoxicity.17-19 Though mechanistically not well understood, one noncytolytic pathway involves a CD8+ T-cell noncytotoxic antiviral T-cell response (CNAR).19 CNAR mitigates viral burden and CD4+ T-cell loss in HIV infection,20-22 but this has not yet been examined in the context of vaccine trials. Thus, vaccine strategies that evoke balanced cytolytic and noncytolytic responses should be explored.23 Such strategies may help impede viral escape arising from mutational inactivation of functional CTL epitopes24,25 and thus require diversified measurements of vaccine-elicited immune outcome beyond the current T-cell response paradigm.
We hypothesized that several immunological functions must converge to limit viral replication and that measuring the ex vivo viral replication (VVR) kinetics on vaccinated monkey peripheral blood mononuclear cells (PBMCs) might provide a way to determine the cumulative effects of different vaccine-induced antiviral mechanisms. Earlier studies with SIV-infected macaques found that susceptibility to infection prechallenge predicted viral load (VL) after challenge.26,27 Resistance to HIV infection of human PBMCs was also increased by vaccine-induced immune responses.28 However, the predictive capacity of ex vivo susceptibility on vaccine outcome has never been tested.
Therefore, we decided to test whether VVR correlated with vaccine-induced protection in the SIV/macaque model. We have compared VVR values with other vaccine-elicited immunological outcomes and viremia after challenge. We show that vaccination suppresses SIV via a noncytotoxic mechanism, and that both CNAR and VVR are associated significantly with preservation of memory CD4+ T cells and control of viremia after challenge.
Experimental Animals and Design
We have extensively described the immunization scheme in our recent publication.29 Briefly, 17 Indian rhesus macaques were divided into 3 groups; 5 in group 1 (sham) and 6 each in groups 2 and 3 which were 3 times DNA primed before twice boosting at 8-week intervals via intramuscular (i.m/systemic) or oral routes. The boosting was done first orally for group 2 (1 × 109 pfu per construct) and intramuscular for group 3 (1 × 108 pfu per construct) at week 24. The second boost was given intramuscular to both vaccine groups at week 32 (2 × 109 pfu/construct). Follow-up lasted 44 weeks. At baseline and every 2-4 weeks during immunization, venous blood was obtained from each animal in citrated vacutainer tubes and used for ficoll gradient preparation of PBMCs.
SIVgag-Specific IFNγ ELISPOT Assays
PBMCs were used to perform IFNγ enzyme linked immunospot (ELISPOT) assays as described in our earlier report.6 Briefly, cells were incubated overnight at 4°C in 96-well plates precoated with anti-IFNγ antibodies, washed with phosphate-buffered saline, and blocked with RPMI-1640, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (R-10) medium. Fifty microliter PBMC suspensions were stimulated with 15-mer SIV gag peptide pools (EVA7066.1-3, CFAR, Program EVA Centralised Facility for AIDS Reagents, Hertfordshire, United Kingdom), control antigens or medium alone. After 16-hour incubation, wash, stain, and detection steps, the plates were air dried and read on a Bioreader-3000 (Bio-Sys GmbH, Karben, Germany). Counts were normalized to negative controls plus input cells and scored as the number of spot-forming units (SFU) per million cells. Positive samples had to yield greater than 100 SFU/106 PBMC above background and twice the medium control signal.
Ex Vivo Infections and Susceptibility Assays
Virus stocks were prepared by infection of CEMx174 cells with SIVmac239-nef-open strain. The infected cells were maintained in R-10 culture medium and harvested on days 6 and 7. These supernatants were pooled, 0.45 μm filter sterilized and aliquots frozen at −80°C. The virus was titrated, and 50% tissue culture infectious dose was determined.30
For VVR assays, PBMCs were stimulated for 24 hours with 10 μg/mL Con-A before infection with SIVmac239 stock at a MOI of 0.003. Based on extensive tests to establish and standardize this method, this multiplicity of infections (MOI) gave highly reproducible results and showed a high dynamic range in VVR values between different animals. The cells were maintained at a density of 2 million per milliliter in RPMI-containing 100 U/mL interleukin-2 (PeproTech Inc, Rock Hill, NJ), 20% FBS, and 1% penicillin/streptomycin (R-20+ medium) for 2 weeks at 37°C in a carbon dioxide chamber. Cultures were supplemented with fresh R-20+ medium every 2-3 days. On 5, 7, and 10 days post ex vivo infection, culture supernatants were aliquoted and frozen at −80°C for subsequent assays.
RNA Extraction and Real-Time Polymerase Chain Reaction
Viral RNA was isolated from 140 μL of freshly thawed culture supernatants and postchallenge plasma using QiaAmp RNA mini spin columns (Qiagen, Hilden, Germany) following manufacturer's protocol. Eluted viral RNA was quantified using TaqMan-based real-time polymerase chain reaction on an ABI-Prism 7500 (Applied Biosystems, Darmstadt, Germany). Primers and standards used for the polymerase chain reaction have been described elsewhere.31 Amplified RNA was expressed as SIV RNA copies per millilitre supernatant or plasma.
Days 7 and 10 PBMC culture supernatants from 26 and 44 weeks during immunization were depleted of the virus by centrifugation at 14,000 rpm and 4°C for 1 hour. The supernatant was screened and found to be negative for viral RNA and infectious virus. Thus, treated supernatant (ΔSUP) was aliquoted and frozen at −20°C until needed. For the assay, TZM-bl cell lines expressing β-galactosidase (provided by Prof Kirchhoff, University of Ulm; originally from National Institutes of Health) were plated onto 96-well flat-bottomed culture plates (Sarstedt, Newton, NC) for 24 hours at a density of 9000 cells per well in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS + 1% penicillin/streptomycin (DMEM+). Before infection, a cocktail comprising SIVmac239 stock diluted in DMEM+ was preincubated with 60 μL ΔSUP for 60-90 minutes at 37°C in a carbon dioxide chamber. To this was added diethylaminoethyl-dextran (DEAE-dextran) just before infection to achieve a final MoI of 0.056, which gave optimum countable spots in the 48 time frame of the test. Positive controls were similarly prepared but with DMEM+ instead of ΔSUP and cells plus medium as negative control. ΔSUP added to the cells acted as background. Upon discarding the medium, 50 μL of the final infection cocktail was added to respective test wells in duplicate or in multiples of 8 for control wells and allowed to adsorb for 2 hours at 37°C before adding 150 μL DMEM+. The plates were incubated for another 42 hrs and stained in the presence of X-gal (CAT# 3522; https://www.aidsreagent.org/reagentdetail.cfm?t=cell_lines&id=34). Dark blue-stained cells were counted using a Bioreader-3000, normalized to medium control, and expressed as infectious SIV titre per millilitre. The titre was converted to percentage inhibition as described elsewhere.32
FACS Analyses for CD4+ Memory T Cells
Citrated blood samples were stained with pretitrated antibody mix comprising anti-CD3-Alexa700 (clone SP34-2), CD4-Alexa405 (SK3), CD8-AmCyan (SK1), CD95-APC (DX2) CD28 PerCP-Cy5.5 (L293), CD25 PE-Cy7 (M-A251), HLA-DR (L243) (Becton Dickinson, Heidelberg, Germany), CD29-FITC (4B4), and CD45RA-ECD (2H4) (Beckman Coulter, Krefeld, Germany) antibodies. After RBC lysis and fixation, samples were measured on a BD LSRII flow cytometer. Data were analyzed using FlowJo.
VVR and plasma VL were log transformed before analysis. Between-group variations were analyzed using Mann-Whitney statistics and longitudinal variations by paired t test. Associations were determined by Pearson correlations and regression analyses. Significance was based on P value ≤0.05.
Animals were housed at the German Primate Centre facility in Goettingen, Germany, and study was conducted according to the German and European Union guidelines on use of nonhuman primates for biomedical research.
VVR Is Reproducible and Predicts VL In Vivo
We first determined if VVR kinetics differed between individual animals and if individual VVR remained stable over time. Polyclonally stimulated PBMCs were infected and maintained in culture. Virus load in culture supernatants was followed from day 5 through day 14 of infected cultures for each animal. We found interindividual VVR variations of >430-fold on day 7 and >60-fold on day 10 after in vitro infection of PBMCs from 17 monkeys (Fig. 1A). The time point of peak VL occurred at day 10 in greater than 90% of individual cultures. Five animals were then tested repeatedly. Their individual VVRs remained stable more than a period of 11 months (Fig. 1B), asserting invariable intrinsic differences in susceptibility of blood cells to SIV infection. Because of a slightly greater stability over time of individual VVR when measured at day 10, we are using the values of this time point throughout the article. However, VVR measured at day 5 or day 7 gave similar results and correlations. When these animals were later infected with SIVmac239, their day 10 VVR correlated significantly with peak VL at 2 weeks postinfection (Fig. 1C) and with VL during the postacute phase (8-16 weeks postinfection). Thus, the individual capacity of PBMCs from vaccine-naive animals to support viral replication ex vivo paralleled susceptibility to infection in vivo.
Because DNA priming followed by Ad 5 vector boosting has been proven to induce strong immune responses and lower VL upon challenge in preclinical AIDS vaccine efficacy studies,6,33 we used a modified approach to evaluate VVR as correlate of protection using 17 monkeys from our parallel immunization study.29 The 5 group 1 animals were sham immunized and acted as controls, whereas groups 2 and 3 received DNA priming and rAd5-SIV boosting as described in Methods. No demonstrable IFNγ ELISPOT responses were detected after just 2 DNA priming or in group 2, 2 weeks after mucosal boosting (Fig. 2A). In contrast, the systemic immunization of group 3 led to peak IFNγ responses 2 weeks after the first boost, which were significantly above those of sham vaccinated or group 2 (P < 0.006). These responses were reboosted to comparable levels after the second rAd5-SIV administration but waned thereafter until the day of challenge (DoC). The IFNγ ELISPOT responses of group 2 peaked after the second boost administered systemically and were still sustained at increased levels on the DoC (Fig. 2A). In these group 2 animals, no marked increase in INFγ ELISPOT levels were observed after oral Ad5 boosting alone.
Intrinsic Susceptibility to SIV Ex Vivo Is Altered by Immunization
We then assessed the influence of our immunization strategy on VVR. Before immunization, animals had been assigned to the 3 study groups. Those with high or low preimmunization VVR were evenly distributed between groups resulting in comparable mean baseline (week 0) VVRs (Fig. 2C). Animals carrying the major histocompatibility complex (MHC-I) allele Mamu-A*01 (in diamonds, Fig. 2B) and known to be associated with strong antiviral CTL responses34 had been evenly assigned to each group. VVRs of these animals did not differ significantly from VVR of animals negative for these alleles. But VVR declined significantly in all vaccines between 0 and 12 weeks after 2 DNA immunizations (P = 0.001, Fig. 2C). At 26 weeks, 2 weeks after rAD5/SIV immunization, VVR was further attenuated in group 3 animals (P = 0.001) boosted systemically (Fig. 2C). VVR was not measured immediately after the second boost (36 weeks during immunization) due to insufficient material. By DoC at 44 weeks during immunization, VVR was attenuated further when compared with baseline values in group 2 animals now boosted systemically (P = 0.011). Compared with sham-vaccinated animals, these kinetics translated into significantly lower VVR for group 2 (P = 0.028) on DoC and group 3 at both week 26 (P = 0.006) and on DoC at 44 weeks during immunization (P = 0.045). As immunization could have influenced the availability of CD4+ T cells as potential targets of in vitro infection, we have analyzed the proportion of different lymphocyte subsets but found neither a difference between unimmunized and immunized animals nor a correlation with VVR.
Vaccine-Induced Correlates of VVR Suppression
We next aimed at delineating the potential immunological effector functions mediating VVR suppression. With the exception of VVR attenuation occurring at 12 weeks during immunization after just 2 DNA immunizations, but without evident increase in INFγ ELISPOT levels, all the other VVR attenuations observed after Ad5 boosting coincided with significantly raised INFγ ELISPOT measurements (Fig. 2A, C). Correlation analyses showed that both VVR and INFγ-secreting cells were significantly associated when measured at 26 weeks during immunization (Fig. 2D), but this did not hold up on DoC (data not shown).
We then asked whether vaccination suppressed VVR via soluble factors. Compared with naive controls, preincubation of virus inoculum with days 7 and 10 culture supernatants from the VVR assays resulted in significant suppression of SIV replication in TZM-bl/CCR5/β-gal indicator cells. Increased suppression was found at 26 weeks during immunization for group 3 (P = 0.028) and for both group 2 (P = 0.006) and group 3 (P = 0.013) animals on DoC (Fig. 3A). This CNAR was inversely associated with VVR both at 26 weeks during immunization (P = 0.014) and on DoC (Fig. 3B). However, the number of INFγ-secreting cells was not associated with CNAR (data not shown), suggesting 2 independent activities. Assuming a linear dose response, we computed regression analyses to determine the extent of VVR dependence on either the INFγ-secreting cells or CNAR. When combined at 26 weeks during immunization, the INFγ response and CNAR contributed up to 67% (R2 = 0.674, P = 0.001) of antiviral activity in vitro. Independently, IFNγ-secreting cells contributed 58% (R2 = 0.579, P = 0.001) and CNAR 41% (R2 = 0.405, P = 0.011) of this activity. On DoC, only CNAR but not IFNγ significantly influenced VVR.
Immunization-Induced Changes in VVR Predict Plasma Viremia and Preservation of Memory CD4+ T Cells After Challenge
We then examined whether the ex vivo infection model was predictive of plasma viremia postinoculation with pathogenic SIVmac239. Vaccination resulted in significant reduction of acute viremia (Fig. 4A, P = 0.002). This effect did not last beyond 12 weeks postchallenge. At this time in the control group, 1 rapid progressor with high viremia had to be sacrificed. This loss narrowed the difference in viremia between control and vaccinated animals during the chronic phase. Since DoC is a realistic baseline for infection studies, we correlated the individual VVRs measured in cell cultures on DoC with plasma viremia after challenge. Individual levels of viral replication ex vivo predicted peak viremia after challenge (Fig. 4B). This association was still observed during postacute phase of infection (P = 0.01 for 8 weeks postchallenge).
Preservation of T-cell memory predicts the outcome of HIV/SIV infection.35 Compared with controls, all vaccinated animals significantly preserved CD29+, CD95+, and memory CD4+ T cells (Fig. 4D, P = 0.03). Individual VVR levels on DoC predicted changes of memory cells in the acute (Fig. 4E) and postacute (P = 0.023) phase after challenge.
Prechallenge CNAR but Not IFNγ Response Is Associated With Plasma Viremia and Memory CD4+ T Cells
Having shown that VVR was dependent partly on CNAR and IFNγ and that VVR predicted plasma viremia and memory CD4+ T-cell levels after challenge, we examined the effects of prechallenge CNAR and the number of INFγ-secreting cells on viremia control and memory CD4+ T-cell levels postchallenge. Strong CNARs occurring on DoC were significantly associated with control of acute plasma viremia (Fig. 4C) and preservation of acute phase memory T cells (Fig. 4F) after challenge. Significant associations were also evident when CNAR activities measured at 26 weeks during immunization were correlated with acute (P = 0.017) and postacute (P = 0.036) memory CD4+ T-cell numbers or with acute (P = 0.011) and postacute (P = 0.042) viremia after challenge. On the contrary, prechallenge numbers of INFγ-secreting cells did not correlate with either memory CD4+ T-cell proportions or plasma viremia.
We have compared the intrinsic ex vivo susceptibility to SIV infection (VVR) before challenge, with vaccine-induced immune responses, memory CD4+ T cells, and plasma viremia after challenge. We have developed this particular in vitro model to comprehensively assess vaccine-induced cellular effector functions. These comparisons allowed us to predict in vivo vaccine protection from the VVR kinetics before challenge of the rhesus macaques.
Intrinsic Susceptibility of Vaccine-Naïve Macaque PBMCs to SIV Infection
Using SIV RNA load in ex vivo infected PBMC cultures as an index for VVR, we observed highly varied viral replication rates between individual vaccine-naive animals. These VVRs of naive animals were constant over time but varied widely between animals, confirming that susceptibility to infection was an individual intrinsic property.27 Although the MHC class-1 genotype is known to affect viremia, CD4+T-cell levels, or disease course,34,36,37 the highly virus restrictive MAMU-A*01 or B*17 alleles did not significantly affect VVR, suggesting that intrinsic susceptibility was unaffected by MHC background. However, it is reasonable to assume that vaccine effects on VVR should be dependent on the MHC genotype, but the low number MAMU-A*01 positive animals precludes a definitive conclusion.
Vaccination Mitigates Ex Vivo Susceptibility to Infection
After vaccination, significant inverse associations were observed between VVR and either the INFγ response or soluble mediators of VVR suppression (CNAR) 2-4 weeks after systemic rAD5/SIV boosting (group 3). This pattern was less evident after oral boosting alone, possibly because oral rAD5/SIV administration alone did not deliver an effective boosting. Overall, systemic boosting resulted in increased CNAR levels still measurable several weeks after immunization on DoC. These CNAR activities correlated with lower VVR, whereas INFγ-secreting cells were low by this time. The short-term stimulation of cells in the IFNγ-ELISPOT assay allows only for measurement of effector cells, which are abundant shortly after immunization but wane afterwards. On the other hand, it is likely that activation of virus-specific memory cells during several days of culture contributes to VVR suppression and CNAR production long after the last immunization. Although the beneficial effect of CNAR has been reported in HAART-treated HIV-infected individuals,21,22 our study provides the first evidence that vaccination significantly raised noncytotoxic antiviral response contributing to virus suppression independent of INFγ-secreting cells. A study in which monkeys were vaccinated with a nef-deleted live virus and challenged with a heterologous strain revealed significant correlations between INFγ and CD8+ T cell-dependent antiviral activity.38 Despite the possibility of this association, in our study, the 2 seemed to influence viral replication more efficiently when statistically assessed independently. Virus suppression does not always depend on cytokine-producing T cells,16 and our data suggest that virus replication, both ex vivo and in vivo, is suppressed by additional hitherto unidentified factors.
Intrinsic Susceptibility Predicts Viremia and CD4+ T-Cell Memory After Challenge
In our study, the extent and duration of protection was similar to comparable DNA prime, Ad5 boost regimens.39,40 In contrast to the study by Casimiro et al4,40, we even found significantly decreased peak and postacute VL and preserved memory CD4 cells not only in MaMuA1*01-positive animals but also in animals negative for this MHC allele.29 Later than 12 weeks postinfection, the difference in these parameters between immunized and unimmunized animals lost the statistical significance, as the first animals of the control group were lost for follow-up because they developed AIDS. An actually longer protection by our vaccination is exemplified by the fact that 7 of 12 immunized animals are still alive at about 3 years after challenge, whereas only 2 of the 6 control animals, both of which carry the long term non-progressors (LTNP)-associated MHC alleles, MaMuA1*01 and MaMuB*17, remained symptom free. In this setting, reduced VVR at DOC in the immunized groups correlated better with postchallenge VL and preservation of memory cells than INFγ responses measured by ELISPOT. Recently, polyfunctional T cells producing multiple cytokines have been established as indicators for vaccine efficacy and susceptibility to infection.9-11 As the paucity of material did not permit to additionally perform intracellular cytokine staining, we can only speculate about the relation between polyfunctionality of T cells and their ability to reduce viral replication in vitro. Also, which of these methods provides the better marker of protection remains to be determined in a side-by-side comparison.
We showed that unimmunized and immunized macaques whose PBMCs replicated the virus less efficiently ex vivo also maintained significantly lower viremia after challenge and better preserved their memory CD4+ T-cell subsets in vivo than high replicators. The characteristic increase in T-cell memory of vaccines was possibly the result of reduced viral burden or a reflection of vaccine-induced expansion of virus-specific cells.35,39,41 Although a limited number of studies have examined the predictive capacity of in vitro viral replication for progression after challenge,26,27 similar studies have not been conducted in immunized monkeys. Likewise, the preservation of memory T-cell subsets is associated with a better prognosis of HIV/SIV infection in vivo,35,39 but whether preservation of these T-cell subsets is already predefined before infection could not been examined. We have shown that VVR on DoC was highly predictive of plasma viremia and memory T-cell levels postchallenge in our immunization protocol. This VVR also offered a better indication of vaccine efficacy than IFNγ. Our data provide the first evidence that individual susceptibility to infection prechallenge predicts loss or preservation of CD4+ T-cells after challenge.
Prechallenge Immune Correlates of Postchallenge Viremia and CD4+ T Cells
Since VVR predicted postchallenge outcomes, we examined how the various ex vivo correlates of VVR-affected viremia and memory T-cell postchallenge. Although we observed no significant associations of prechallenge INFγ ELISPOT results with plasma viremia or memory T-helper cells, CNAR was highly predictive of both postchallenge outcomes during the acute through chronic phase of infection. These latter associations could be attributed to sustained production of soluble factors rather than cytolytic activity. During viral latency, a critical microenvironment sustains low-level infection.42,43 This microenvironment provides a survival advantage via an alternative innate antiviral mechanism before restoration of virus-specific cytotoxic T-cell responses. We postulate that postacute and chronic phase SIV infection is influenced by noncytotoxic antiviral activities, which may play a major role in limiting viral replication and facilitating recovery of virus-specific immune responses. Vaccine designs that ensure sustained noncytotoxic antiviral responses may improve robustness and efficacy. Additional effort should focus on detailed characterization of the mediators of VVR suppression in animal models and human elite controllers.
In conclusion, we show that this ex vivo challenge model allows for evaluation of vaccine potency before challenge because it accurately predicts the in vivo virus replication and CD4+ T-cell levels. In our setting, outcome of infection after challenge was better predicted by VVR than by IFNγ ELISPOT, a commonly used measure of the cellular immune response. This test platform offers an opportunity for human AIDS vaccine trials, in which volunteer PBMCs could be used to rapidly determine the efficacy of candidate vaccines in the absence of infection exposure. Such an approach would spare time and financial, human, and animal resources and circumvent ethical and design constraints.
We thank the EU's Programme EVA and the Centralised Facility for AIDS Reagents for providing SIV peptides.
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