Early potent protection against heterologous SIVsmE660 challenge following live attenuated SIV vaccination in Mauritian cynomolgus macaques

Abstract

Background: Live attenuated simian immunodeficiency virus (SIV) vaccines represent the most effective means of vaccinating macaques against pathogenic SIV challenge. However, thus far, protection has been demonstrated to be more effective against homologous than heterologous strains. Immune correlates of vaccine-induced protection have also been difficult to identify, particularly those measurable in the peripheral circulation.

Methodology/principal findings: Here we describe potent protection in 6 out of 8 Mauritian-derived cynomolgus macaques (MCM) against heterologous virus challenge with the pathogenic, uncloned SIVsmE660 viral stock following vaccination with live attenuated SIVmac251/C8. MCM provided a characterised host genetic background with limited Major Histocompatibility Complex (MHC) and TRIM5α allelic diversity. Early protection, observed as soon as 3 weeks post-vaccination, was comparable to that of 20 weeks vaccination. Recrudescence of vaccine virus was most pronounced in breakthrough cases where simultaneous identification of vaccine and challenge viruses by virus-specific PCR was indicative of active co-infection. Persistence of the vaccine virus in a range of lymphoid tissues was typified by a consistent level of SIV RNA positive cells in protected vaccinates. However, no association between MHC class I/II haplotype or TRIM5α polymorphism and study outcome was identified.

Conclusion/significance: This SIV vaccine study, conducted in MHC-characterised MCM, demonstrated potent protection against the pathogenic, heterologous SIVsmE660 challenge stock after only 3 weeks vaccination. This level of protection against this viral stock by intravenous challenge has not been hitherto observed. The mechanism(s) of protection by vaccination with live attenuated SIV must account for the heterologous and early protection data described in this study, including those which relate to the innate immune system.

Figure 1. Infectivity of SIVsmE660 challenge stock in Mauritian cynomolgus macaques.

Levels of plasma SIV RNA measured by qRT-PCR with R/U5 primers following multiple SIVsmE660 challenges in an in vivo titration of unvaccinated Mauritian cynomolgus macaques. Red arrow shows point of SIVsmE660 inoculation (day 0). Plasma SIV RNA levels are shown for individual macaques (B1–B8) and (B95–B100) in two independent titration series (panels A and B respectively) comparing acute, peak and steady-state chronic phases of virus replication post-SIVsmE660 challenge. Outcome of administering high dose (low dilution) viral inocula for pairs of macaques receiving a 1/10 (Group 1 ; B1, B2), 1/100 (Group 2; B3, B4); 1/1000, (Group 3, B5, B6), and 1/10,000 (Group 4, B7, B8) dilution of virus are shown in panel A. Plasma viraemia for the second dilution series at 1/10,000 (Group 5; B95, B96), 1/100,000 (Group 6; B97, B98) and 1/1,000,000 (Group 7, B99, B100) dilutions are shown in panel B. No evidence of plasma virus was detected in B97, B99 and B100 at any time point.

Figure 2. Distribution of MHC haplotypes in 26 Mauritian cynomolgus macaques.

MHC genotype distribution for the in vivo titration of SIVsmE660 (see Figure 1) in macaques B1–B8 and B95–B100 (panel A). Twelve macaques (B202–B213) were included in the vaccine/challenge study (panel B). Distribution of M1–M6 haplotypes for Class IA, Class IB and Class II for each animal is shown in pictorial format represented as: M1 (black); M2 (red); M3 (blue), M4 (green), M5 (yellow) and M6 (grey). No M7 haplotype was identified. Recombinants are represented as multiple colours.

Figure 3. Vaccine virus kinetics.

Kinetics of virus replication post-SIVmacC8 vaccination measured by SIVmac251/C8-specific quantitative RT-PCR in 20 weeks (Group A) and 3 weeks (Group B) vaccinates. Blue arrow shows point of SIVmacC8 inoculation, red arrow point of SIVsmE660 challenge. Differences in vaccine kinetics between vaccinates in Panel A, Group A (B202–B205), after the peak of virus replication was seen, prior to SIVsmE660 challenge. Panel B, shows a single time-point 21 days post SIVmacC8 inoculation, ie day of SIVsmE660 challenge. In both Groups A and B, recrudescence of the vaccine virus SIVmacC8 upon SIVsmE660 challenge is shown.

Figure 4. Anti-gp130 levels.

Anti-gp130 levels post-SIVmacC8 vaccination and post SIVsmE660 challenge determined by binding antibody ELISA, (day 0 represents day of SIVsmE660 challenge) shown for Groups A, B and C. Group A shows anti-SIVmacC8 gp130 levels for 20 weeks prior to SIVsmE660 challenge (at day 0) with an anamnestic response detected in B204. Panel B shows anti-gp130 responses in Group B (3 week vaccinates) and Group C (SIVsmE660 naïve challenge controls) respectively.

Figure 5. CD4 lymphocyte percentages.

CD4 lymphocyte percentages are shown for 20 week SIVmacC8 vaccinates (Group A; panel A) or 3 weeks (Group B; panel B), following SIVsmE660 challenge and unvaccinated challenge controls (Group C; panel C). Timings of all bleeds were taken from initial time of SIVsmE660 challenge (day 0). Group mean values with SE of the mean values are shown in panel D.

Figure 6. Total SIV RNA levels in plasma.

SIV RNA levels, expressed as log₁₀ SIV RNA copies/ml, determined by virus-common qRT-PCR assay based on conserved R/U5 sequences in genomic SIV RNA. Panel A, 20 weeks vaccinates (Group A; B202–B205); panel B, 3 weeks vaccinates (Group B; B206–B209); panel C, 9 naive controls challenged with low dose (1–10MID₅₀) SIVsmE660.

Figure 7. Type common and type specific PCR analysis of SIVmac251 and SIVsmE660.

Panel A shows equivalent amplification of SIVmac251 and SIVsmE660 with R/U5 primers targeting the 5′ region of genomic SIV RNA. Comparable regression curves were obtained with a high titre SIVmac251 reference panel and a SIVsmE660 plasma pool derived from day 10 and 14 bleeds of titration macaques B1–B4. Subsequent cross-titration experiments conducted with the heterologous gag-based SIVsmE660 and SIVmacC8 plasma viral RNA quantification assays compared threshold detection levels with viral RNA in-put copy number. Assay specificity was demonstrated with the same pooled SIVsmE660 plasma from control MCMs diluted in negative plasma and a SIVmac251/L28 plasma reference panel for SIVmac251 sequences. Respective assays demonstrated specific amplification of the SIVsmE660 (challenge, panel B) and SIVmac251 (vaccine, panel C) viruses by gag-specific RT-PCR across a 6 log₁₀ dynamic range. Intra-assay variation between replicates of the SIVsmE660-specific assay was 0.09 SD with a minimum amplification efficiency of 94.9%. Replicates included at least three runs with a coefficient of variation of <3%. No cross-reactivity with the heterologous virus was detected above a sensitivity of detection limit of 50 SIV RNA copies/ml.

Figure 8. Differential vRNA analysis of SIVsmE660 and SIVmacC8 in plasma.

Plasma SIV RNA levels shown for superinfected vaccinates (B204/B209) and six protected vaccinates (B202, B203, B205, B206, B207, B208) using a SIVsmE660-specific viral RNA assay (panels A and B respectively), compared to unvaccinated naive challenge controls (panel C) with the same assay. Controls were eight naïve MCMs challenged with 10 MID₅₀ of the SIVsmE660 stock (1/10,000 dilution of the original stock; B7, B8, B95, B96, B210–B213) or productively infected at the 1/100,000 dilution (B98). Levels of the vaccine virus SIVmacC8 are shown in panels D and E for superinfected and protected vaccinates respectively with the SIVmac251-specific qPCR assay . Panel F shows no reactivity >50 SIV RNA copies/ml of the SIVmacC8-specific assay with unvaccinated challenge controls sampled at 14, 84 and 140 days post SIVsmE660 challenge.

Figure 9. SIVsmE660 DNA levels in lymphoid tissues.

Levels of SIVsmE660-specific DNA in lymphoid tissue in SIVsmE660 controls and 20 and 3 week SIVmacC8 vaccinates shown according to superinfection or protection status. SIV DNA levels are expressed as copies of SIVsmE660 DNA / 100,000 cell equivalents MNCs. Limit of detection is 1 copy/100,000 cell equivalents MNC DNA.

Figure 10. Distribution of SIV RNA positive cells by ISH in protected/unprotected vaccinates and SIVsmE660 controls.

Panels A and B) Mesenteric lymph node (MLN) sections from 20 and 3 week SIVmacC8 vaccinates (B202 and B207) protected from SIVsmE660 challenge. Panels C and D show spleen (spl) and MLN from 20 and 3 week SIVmacC8 vaccinates subsequently superinfected with SIVsmE660. B204 shows multiple foci of SIV RNA positive cells in spleen. Panels E and F show MLN and small intestine (S.Int) sections of naive challenge control macaques B210 and B213 after 20 weeks SIVsmE660 infection. B213 exhibits occasional clusters of SIV positive cells. Black staining indicates foci of SIV infection as indicated by arrows.