Increased cellular immune responses and CD4+ T-cell proliferation correlate with reduced plasma viral load in SIV challenged recombinant simian varicella virus - simian immunodeficiency virus (rSVV-SIV) vaccinated rhesus macaques

An effective human immunodeficiency virus type 1 (HIV-1) vaccine is needed to control the HIV pandemic. The use of live attenuated viruses as vaccines has demonstrated protection from rigorous homologous and heterologous viral challenges in macaques, thus providing critical proof-of-concept for the feasibility of the development of an effective HIV vaccine to prevent or limit HIV infection[1‐3]. The result of clinical trial in Thailand testing with various subtype B canarypox-HIV-1 recombinant vaccine candidates and boosters containing subunit glycoprotein 120 or 160 did not generate strong cellular or detectable neutralizing antibody responses to HIV-1 yet showed marginally significant protection from infection[4]. A recent study with adenovirus/poxvirus and adenovirus/adenovirus-vector-based simian immunodeficiency virus (SIV) vaccine was shown to block acquisition of pathogenic heterologous, neutralization-resistant challenge virus in the rhesus macaque (RM) model[5]. A similar study with persistent cytomegalovirus (CMV) vector-based SIV vaccine was able to control highly pathogenic SIV infection by inducing effector memory T-cell responses[6]. So far, the development of an effective HIV vaccine that is capable of protecting new infection remains elusive.

Live-attenuated varicella-zoster virus (VZV) Oka vaccines have been shown to be safe and effective for immunization against VZV infection (chickenpox and shingles). This strongly immunogenic herpes virus vaccine backbone provides an attractive candidate for designing recombinant vaccines[7]. Periodic subclinical reactivation of VZV from latency may provide persistent immune re-stimulation to VZV and to foreign antigens. Due to stringent VZV host-range restrictions, the simian counterpart virus, simian varicella virus (SVV), with established RM models of both varicella and AIDS, provide an alternative experimental approach to investigate varicella pathogenesis and AIDS vaccine development[8, 9]. Our recent study using recombinant SVV (rSVV) expressing SIV Gag + Env antigens (rSVV-SIV_MAC239Gag and rSVV-SIV _MAC239Env) demonstrated reduced plasma viral loads (VLs) in five immunized RMs (Experimental Group, EG) following intravenous SIV_MAC251/CX1 challenge, when compared to four Controls vaccinated (rSVV-RSV _G and SVV-RSV _M2 ) RMs (Control Group, CG) challenged with SIV_MAC251/CX1[10]. These results highlight the strengths and success of the SVV model to evaluate SIV/HIV vaccine candidates[10, 11].

The present study investigates the role of cellular immune responses in rSVV-SIV immunized animals to define correlates of protection. All animals (EG and CG) were negative for HIV-2, SIV, type D retrovirus and STLV-1 infection at the beginning of the study. All the animals were housed at the Tulane National Primate Research Center (TNPRC) and under the full care of TNPRC veterinarians in accordance with the standards incorporated in the Guide to the Care and Use of Laboratory Animals (NIH) 78–23 (Revised, 1996). All veterinary procedures were performed only with sedated animals. All animal procedures were reviewed and approved by the Tulane Institutional Animal Care and Use Committee. The levels of T-cell proliferation, memory cell populations, polyfunctional T-cell responses in PBMC were assessed by flow cytometry and their responses were correlated with plasma VLs. In brief, T-cell immunophenotyping and Ki67 staining were performed using anti-CD3-FITC/PerCP (SP34-2), CD4-APC (L200), CD8-PE/PerCP (RPA-T8), CD95-FITC (DX2), CD28-PE (CD28.2) and anti-Ki67-PE (B56) monoclonal antibodies (mAbs) obtained from BD Biosciences (BD) as reported earlier[12]. Data was acquired on a FACS Calibur flow cytometer using BD CellQuest software and analyzed using FlowJo software, version 9.1. (TreeStar Inc., Ashland, OR).

The naïve (CD28 + CD95-) T-cell population increased in both groups following rSVV immunization (Figure 1A), and is possibly due to heightened immune functioning, although neither increase was statistically significant. Following SIV challenge, however, the CG animals continued with a sharp rise from 28.4% on day of challenge (doc) to 35.2% on d14 post-challenge (d14pc) and remained high for the d231pc monitoring period (23%-37%). These findings were significantly different (p < 0.05) and in contrast to the homologous SIV challenged EG animals, with 20% naïve T-cells on doc and continued progressive decline to 8% at d231pc. This decline is likely due to a higher recruitment of naïve cells to SIV specific memory cells in this EG. Conversely, the central memory (CD28 + CD95+) T-cell population was maintained in both groups following immunization. After SIV challenge, a selective depletion of central memory T-cells was observed in both CG [22.9% (doc), 15.3% (d14pc), 10% (d119pc)] and EG [22% (d14pc), 15% (d119pc)] with no statistically significant differences within groups (Figure 1B). Finally, the effector memory (CD28-CD95+) T-cells peaked on d14pc with mean values 22.5% and 28.0%, in CGs and EGs respectively. This cell population decreased in both groups over the next two months to 15% followed by a gradual increase, slightly greater in the EG over the CG, although not statistically significant (Figure 1C). This sustained increase in the effector memory cell population following SIV challenge is thought to be due to the loss of the central memory T-cell population and induction of antiviral functions in plasma viral load reduction in both EG and CG animals.

Next, we analyzed the level of proliferation in CD3+CD4+ T-cells and CD3+CD8+ T-cells to determine the impact of rSVV-SIV vaccination in response to SIV challenge (Figure 2). Following SIV challenge, an initial sharp increase in CD4+Ki67+ proliferating T-cells was observed in both vaccinated groups. The EG mean levels rose from 1.3% (doc) to 3.6% (d28pc) and maintained at significantly higher levels (P < 0.001) for the d231pc. The CG mean rose to 2.2% (d28pc), was transient however, decreased to baseline levels by d84pc and maintained lower than the EG for the remainder of the monitoring period. Similar increases in CD4+Ki67+ cells have also been observed in HIV-1 and pathogenic SIV infections[13‐15]. An immediate increase in CD8+Ki67+ T-cells was observed in both EG and CG that was short-lived and returned to baseline by d28pc, supporting the similar expansion of CD8+ cells in peptide vaccine and SIV pathogenesis studies reported elsewhere[12, 14].

Mean levels of plasma viremia were correlated with percentages of proliferating (Ki67+) CD4+ and CD8+ T-cells for both EG and CGs at each of the time points post SIV challenge (d14pc to d231pc). Pearson coefficient of determination analysis of these independent measures showed a highly significant inverse correlation of the proliferating CD4+Ki67+ T-cells and plasma viremia, regardless of treatment or timepoint (Figure 3). Linear regression analysis of these data resulted in a straight line with a slope 0.92 ± 0.15 (r² = −0.702; p < 0.0001). All correlated values for the CG showed low levels of CD4+Ki67+ T-cells (0.9–2.2%) and high plasma VLs (log^6.3–7.6 copies/ml) while all values but one for the EG had high levels of CD4+Ki67+ T-cells (2.5–4.1%) and low plasma viral loads (log^{4.7 – 5.6} copies/ml). The initial high viremic spike (log^6.8 copies/ml) in the EG at d14pc also correlated with a low level of CD4+Ki67+ T-cells (1.8%) (Figure 3).

To assess the magnitude and functional characteristics of SIV specific CD4+ and CD8+ T-cells from both groups of animals, intracellular cytokine staining was performed with proper positive and negative controls as described earlier[16]. Resuspended PBMCs at 14 days post immunization (d14pi), doc and d231pc time points were stimulated with SIV-Gag (cat. 6204) and Env (cat. 6883) peptide pools (NIH AIDS Research & Reference Reagent Program) in the presence of 0.5ug/ml of anti-CD28 (clone CD28.2, BD) and anti-CD49d (clone 9F10, BD) mAbs. Following stimulation, the cells were stained for live/dead stain (Invitrogen), then surface staining with anti-CD3, anti-CD4, anti-CD8; and intracellular staining with anti-IL2 (MQ1-17H12), anti-IFNγ (4S.B3), and anti-TNFα (MAb11) mAbs. Data acquisition was performed on fixed stained cells using BD LSRII flow cytometer and analyzed using FlowJo software, version 9.1.[16] (Additional file1: Figure S1). The analysis and presentation of cytokine expression was performed for each animal after proper antigenic stimulation and using SPICE software version 5.2, downloaded fromhttp://​exon.​niaid.​nih.​gov/​spice (Figure 4). In brief, cytokine responses produced by macaques are either monofunctional (mono, producing any one cytokine), bifunctional (bi, any combination of two different cytokines), or trifunctional (tri, producing three cytokines) cells and are diagrammatically represented in Figure 4. Recombinant SVV Experimentally immunized macaques (EG) generated SIV-Env specific monoCD4+, (mean values 0.50%, 0.66%, 0.12% on d14pi, doc, d231pc respectively) as well as biCD4+ (0.01%, 0.01%, 0.03% on d14pi, doc, d231pc respectively) (Figure 4, Additional file2: Figure S2, Table 1). SIV-Env specific CD8+ responses were also demonstrated in the rSVV vaccinated EG macaques with monoCD8+ mean value responses of 0.08%, 0.18%, 0.02%; and biCD8+ mean values of 0.01%, 0.03%, 0.0% at d14pi, doc, d231pc respectively. In contrast, Control vaccinated (CG) macaques produced responses for SIV-Env specific cells with results of monoCD4 mean values of 0.0%, 0.0%, 0.19%; biCD4 mean values 0.0%, 0.0%, 0.0%; monoCD8 mean values 0.0%, 0.0%, 0.05%; and biCD8 mean values 0.0%, 0.0%, 0.0% on d14pi, doc, d231pc respectively (Figure 4, Additional file2: Figure S2, Table 1). These data show that all EG macaques demonstrated substantial SIV specific cytokine responses in both CD4 and CD8 cells at pre challenge time points with CD4 responses greater than CD8 cytokine responses. All CG macaques were completely negative for SIV specific CD4+ and CD8+ cytokine responses at those prechallenge time points (Table 1). Postchallenge results showed positive cytokine expression in 4 out of 5 EG animals and 2 out of 4 CG animals (Additional file2: Figure S2). Mean responses were lower in both groups (EG and CG), compared to the prechallenge responses, possibly due to immunosuppression in this late chronic phase of the SIV infection (Table 1).

Although we tested only one timepoint (d14pi) for SIV-Gag specific responses in EG, the values were 1.5-10 times higher than the SIV-Env specific responses at this d14pi time point. EG animals showed SIV-Gag specific monoCD4+ response mean of 0.86%, monoCD8+ mean of 0.43%, biCD4+ mean 0.79% and biCD8+ mean 0.18%, were higher when compared with CG responses for monoCD4+ mean 0.06%, biCD4+ mean 0.05%, monoCD8+ mean 0.05%, and biCD8+ mean 0.01% at d14pi. CG animals only were additionally tested for Gag-specific cytokine responses on doc and d231pc with no detectable responses for any animal in this group (Table 1). These findings demonstrate greater mono and polyfunctional responses during prechallenge time points in the EG over the CG animals and that CD4+ T-cells play a major role in inducing increased cytokine responses compared to CD8+ T-cells. In addition, although only at the one time-point tested, the gag-specific responses were greater than the Env-specific responses in EG animals compared to CG animals.

The increased CD4+ T-cell proliferation and profound SIV-Gag and Env specific cytokine responses in Experimental vaccinated macaques suggest that those proliferating CD4+ T-cells may be effector cells and their SIV-specific effector functions contributed significantly to control plasma VLs. Low to minimal neutralizing antibody responses in those rSVV-SIV vaccinated animals[10] also suggest that SIV-specific cytokine responses may play a crucial role in controlling plasma VLs and disease progression. Vaccine induced increased CD4+ T-cell proliferation and cytokine responses support earlier observations where HIV-specific CD4 T-cells were thought to be responsible for enhanced immunological control of HIV viremia either by helping CD8 T and B cells[17] or by direct antiviral effects[18, 19]. Enhanced HIV-specific CD4 T-cells cytokine responses were also demonstrated in individuals that are able to control viral replication spontaneously in the absence of antiretroviral therapy[20, 21]. Finally, with only a small percentage of HIV-specific CD4+ T-cells preferentially infected by HIV, the vast majority of uninfected CD4+ T-cells would be present and capable of inducing antiviral activity[22].

In conclusion, it is still unclear what constitutes the correlates of protection and what early immune responses are required to prevent early virus dissemination, viral replication, and viral transmission. In an effort to define those correlates, this vaccine study shows that increased CD4+ T-cell proliferation and increased SIV-antigen specific mono and polyfunctional CD4 and CD8 responses in the rSVV-SIV_Env/Gag vaccinated animals are key correlates of vaccine-mediated protection. These results show significant promise for rSVV-SIV vaccines as an effective preclinical approach to test potential recombinant AIDS vaccines with subsequent translation into rVZV-HIV vaccination in humans.