Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine

Abstract

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (T(EM)) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIV(MAC239) infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4(+) memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8(+) T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8(+) or CD4(+) lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated T(EM) responses might significantly contribute to an efficacious HIV/AIDS vaccine.

Figure 1. Immunogenicity and efficacy of RhCMV/SIV vectors

a, Schematic of the vaccination protocol used in this study. b, Comparison of the mean frequency (± SEM) of the overall SIV-specific CD4+ and CD8+ T cell responses and the contribution of the designated SIV proteins to these total responses in the blood memory compartments of Groups A-C RM at the end of the vaccine phase. The Kruskal-Wallis test was used to determine the significance of differences in total SIV-specific response frequencies among the 3 vaccine groups, with the Wilcoxon rank sum test used to perform pair-wise analysis for the CD8+ response. As these latter p values were > 0.05, we concluded that overall response frequencies of Groups A, B and C were not significantly different. c, Outcome of repeated, limiting dose, intra-rectal SIVmac239 challenge of Groups A-D. The significance of differences in the fraction of infected RM in each group that met controller criteria (see Methods) was determined by Fisher's exact test (closed symbols in Group D are concurrent controls; open, previous controls given the same challenge). d, Analysis of the magnitude and frequency of pvl “blips” in Group A and B controllers, with the significance of the differences in blip magnitude and frequency between Groups A and B determined by the Wilcoxon rank sum test, and the significance of the decline in blip frequency of Group A + B RM after 30 weeks pi determined by analysis of variance and linear trend tests. e, Comparison of pvl in Group A-D RM with progressive infection (excluding Group A and B controllers and Group D RM with protective MHC alleles not represented in Groups A-C) with the significance of differences between Groups A, B and C vs. Group D determined by the Wilcoxon rank sum test.

Figure 2. Immunologic correlates of RhCMV/SIV vector-associated control

a, Analysis of total SIV-specific CD8+ T cell responses in the blood memory compartment during the vaccine phase of Group A and B RM with differences in the magnitude of these responses between controllers and non-controllers at the designated time points determined by the Wilcoxon rank sum test. b, Comparison of the anti-SIVenv antibody titres in plasma (as measured by neutralization of tissue culture-adapted SIVmac251) before and following infection of controller vs. non-controller RM among Group A-C and the concurrent Group D RM (RP = rapid progressor). The significance of the differences in log change in Ab titre from pre-infection to day 112 pi in Group A and B controllers vs. Group C RM was determined by the Wilcoxon rank sum test. c, Analysis of the change in the SIVgag-specific CD4+ and CD8+ T cell response frequency following controlled vs. progressive infection in Groups A, B and C with the significance of differences in peak response boosting between the designated groups determined by the Wilcoxon rank sum test.

Figure 3. Immunologic characterization of long-term control associated with RhCMV/SIV vector vaccination

a, Analysis of the effect of depletion of CD8+ lymphocytes with mAb cM-T807 on viral replication and boosting of SIVvif-specific T cell responses (in the non-depleted CD4+ subset) in 4 long-term RhCMV/SIV vector-vaccinated controllers (2 Group A and 2 Group B RM) vs. 2 conventional controllers (1 Group C, DNA/Ad5-vaccinated controller; 1 Group D spontaneous controller). b, Analysis of the frequencies of blood CD8+ T cells specific for SIV proteins that were (gag, pol) or were not (vif) included in the CMV/SIV vectors in the 4 Group A “controllers” for which long-term data is available. The response frequencies were normalized to the response frequencies immediately prior to SIV infection for the gag- and pol-specific responses, and to the peak frequencies following SIV infection for the vif-specific responses. The 4 RM used in this long-term response analysis include those subjected to transient CD4+ or CD8+ lymphocyte depletion (2 each). As Ag-specific CD8+ responses cannot be reliably determined during the period of overall CD8+ lymphocyte depletion, these periods are shown as gaps for 2 affected RM. The significance of differences in the maintenance of response frequencies of gag- and pol- vs. vif-specific CD8+ T cells in these RM was determined by Wilcoxon rank sum analysis.

Figure 4. Measurement of SIV RNA and DNA in long-term RhCMV/SIV vector-vaccinated controllers

Nested qPCR and qRT-PCR analysis of SIV DNA and RNA, respectively, on tissue obtained at necropsy from an uninfected RM, 4 long-term (>52 wk) RhCMV/SIV vector-vaccinated controller RM (1 Group A; 3 Group B), 2 conventional controller RM [a live attenuated SIV (LAV)-vaccinated RM that resisted wildtype SIVmac239 challenge 33 and 10 weeks prior to necropsy, and a Group C, DNA/Ad5 vaccine-protected RM at 55 weeks pi], and an RM with poorly controlled SIV infection [a LAV-vaccinated RM 24 weeks after wildtype SIVmac239 challenge]. Filled square plot symbols indicate DNA or RNA copy numbers based on directly measured values for samples giving 10/10 replicate reactions positive. Filled triangles indicate results for samples giving at least one, but <10, replicate reactions positive, with copy number imputed by Poisson distribution. Open circles indicate specimens that gave 0/10 replicates positive with the symbol's position in the plots at the threshold value corresponding to a Poisson distribution imputed copy number corresponding to 1/10 replicates positive. All values are normalized for nucleic acid input. Arrowheads indicate the highest threshold value for negative samples (0/10 replicates positive) for all of the tissues analyzed for that RM. “GI Tract and Associated LN” include colon/rectum, ileum, jejunum, superior/medial/inferior mesenteric and ileocecal LNs. “Peripheral LN” include axillary, submandibular, inguinal, iliosacral, and tracheobronchial LNs. “Other Hematolymphoid Tissues” include liver, spleen, bone marrow, tonsil, and thymus.