Search

Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication-defective adenovirus (rAd) vaccine vectors are at the forefront of this work and have been tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV Gag or other nonenvelope proteins induces T cell responses that control virus load but disappointingly is unsuccessful so far in preventing infection, and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomolgus macaques (MCM), Macaca fascicularis , vaccinated with unmodified SIV gag alone in a DNA prime followed by an rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low-dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection, a significant reduction in peak virus load was observed in all vaccinated animals, including those vaccinated with modified gag These findings suggest that a nonpersistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV type 1 (HIV-1) infection. The mechanisms are probably distinct from those of antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus-infected cells and may be mediated in part by CD4 T cells. IMPORTANCE The simian immunodeficiency virus (SIV) macaque model represents the best animal model for testing new human immunodeficiency virus type 1 (HIV-1) vaccines. Previous studies employing replication-defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with an rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to the envelope and neutralize the virus or mediate antibody-dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established has also shown some success. In contrast, the vaccine described here mediates resistance by a different mechanism from that described above, which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development., (Copyright © 2019 Almond et al.)

In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity., Competing Interests: The authors have declared that no competing interests exist.

Vaccination with live attenuated simian immunodeficiency virus (SIV) in non-human primate species provides a means of characterizing the protective processes of retroviral superinfection and may lead to novel advances of human immunodeficiency virus (HIV)/AIDS vaccine design. The minimally attenuated SIVmacC8 vaccine has been demonstrated to elicit early potent protection against pathogenic rechallenge with genetically diverse viral isolates in cynomolgus macaques (Macaca fascicularis). In this study, we have characterized further the biological breadth of this vaccine protection by assessing the ability of both the nef-disrupted SIVmacC8 and its nef-intact counterpart SIVmacJ5 viruses to prevent superinfection with the macrophage/neurotropic SIVmac239/17E-Fr (SIVmac17E-Fr) isolate. Inoculation with either SIVmacC8 or SIVmacJ5 and subsequent detailed characterization of the viral replication kinetics revealed a wide range of virus-host outcomes. Both nef-disrupted and nef-intact immunizing viruses were able to prevent establishment of SIVmac17E-Fr in peripheral blood and secondary lymphoid tissues. Differences in virus kinetics, indicative of an active process, identified uncontrolled replication in one macaque which although able to prevent SIVmac17E-Fr superinfection led to extensive neuropathological complications. The ability to prevent a biologically heterologous, CD4-independent/CCR5+ viral isolate and the macrophage-tropic SIVmac316 strain from establishing infection supports the hypothesis that direct target cell blocking is unlikely to be a central feature of live lentivirus vaccination. These data provide further evidence to demonstrate that inoculation of a live retroviral vaccine can deliver broad spectrum protection against both macrophage-tropic as well as lymphocytotropic viruses. These data add to our knowledge of live attenuated SIV vaccines but further highlight potential safety concerns of vaccinating with a live retrovirus.

Vaccination of Mauritian cynomolgus macaques with the attenuated nef-truncated C8 variant of SIVmac251/32H (SIVmacC8) induces early, potent protection against pathogenic, heterologous challenge before the maturation of cognate immunity. To identify processes that contribute to early protection in this model the pathogenesis, anatomical distribution and viral vaccine kinetics were determined in relation to localised innate responses triggered by vaccination. The early biodistribution of SIVmacC8 was defined by rapid, widespread dissemination amongst multiple lymphoid tissues, detectable after 3 days. Cell-associated viral RNA dynamics identified mesenteric lymph nodes (MLN) and spleen, as well as the gut mucosae, as early major contributors of systemic virus burden. Rapid, localised infection was populated by discrete foci of persisting virus-infected cells. Localised productive infection triggered a broad innate response, with type-1 interferon sensitive IRF-7, STAT-1, TRIM5α and ApoBEC3G genes all upregulated during the acute phase but induction did not prevent viral persistence. Profound changes in vaccine-induced cell-surface markers of immune activation were detected on macrophages, B-cells and dendritic cells (DC-SIGN, S-100, CD40, CD11c, CD123 and CD86). Notably, high DC-SIGN and S100 staining for follicular and interdigitating DCs respectively, in MLN and spleen were detected by 3 days, persisting 20 weeks post-vaccination. Although not formally evaluated, the early biodistribution of SIVmacC8 simultaneously targets multiple lymphoid tissues to induce strong innate immune responses coincident at the same sites critical for early protection from wild-type viruses. HIV vaccines which stimulate appropriate innate, as well as adaptive responses, akin to those generated by live attenuated SIV vaccines, may prove the most efficacious.

Background: Live attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV., Results: Using a novel, doxycycline dependent, replication-competent live-attenuated SIVmac239Δnef (SIV-rtTAΔnef), we show that under replication-permissive conditions SIV-rtTAΔnef is fully viable. Twelve rhesus macaques were infected with a peak plasma vRNA on average two log10 lower than in 6 macaques infected with unconditionally replication-competent SIVΔnef. Consistent with the attenuated phenotype of the viruses the majority of animals displayed low or undetectable levels of viraemia by 42-84 days after infection. Next, comparison of circulating T cells before and after chronic infection with parental SIVΔnef revealed a profound global polarisation toward CD28-CCR7- T-effector memory 2 (TEM2) cells within CD95+CD4+ and CD95+CD8+ populations. Critically, a similar effect was seen in the CD95+ CD4+ population and to somewhat lesser extent in the CD95+ CD8+ population of SIV-rtTAΔnef chronically infected macaques that were maintained on doxycycline, but was not seen in animals from which doxycycline had been withdrawn. The proportions of gut-homing T-central memory (TCM) and TEM defined by the expression of α4β7 and CD95 and differential expression of CD28 were increased in CD4 and CD8 cells under replication competent conditions and gut-homing CD4 TCM were also significantly increased under non-permissive conditions. TEM2 polarisation was seen in the small intestines of animals under replication permissive conditions but the effect was less pronounced than in the circulation. Intracellular cytokine staining of circulating SIV-specific T cells for IL-2, IFN-γ, TNF-α and IL-17 showed that the extent of polyfunctionality in CD4 and CD8 T cells was associated with replication permissivity; however, signature patterns of cytokine combinations were not distinguishable between groups of macaques., Conclusion: Taken together our results show that the global T memory cell compartment is profoundly skewed towards a mature effector phenotype by attenuated SIV. Results with the replication-conditional mutant suggest that maintenance of this effect, that may be important in vaccine design, might require persistence of replicating virus.

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.

APOBEC3G is an innate intracellular anti-viral factor which deaminates retroviral cytidine to uridine. In vivo studies of APOBEC3G (A3G) were carried out in rhesus macaques, following mucosal immunization with SIV antigens and CCR5 peptides, linked to the 70kDa heat shock protein. A progressive increase in A3G mRNA was elicited in PBMC after each immunization (p<0.0002 to p< or =0.02), which was maintained for at least 17 weeks. Analysis of memory T cells showed a significant increase in A3G mRNA and protein in CD4(+)CCR5(+) memory T cells in circulating (p=0.0001), splenic (p=0.0001), iliac lymph nodes (p=0.002) and rectal (p=0.01) cells of the immunized compared with unimmunized macaques. Mucosal challenge with SIVmac 251 showed a significant increase in A3G mRNA in the CD4(+)CCR5(+) circulating cells (p<0.01) and the draining iliac lymph node cells (p<0.05) in the immunized uninfected macaques, consistent with a protective effect exerted by A3G. The results suggest that mucosal immunization in a non-human primate can induce features of a memory response to an innate anti-viral factor in CCR5(+)CD4(+) memory and CD4(+)CD95(+)CCR7(-) effector memory T cells.

Vaccination with live attenuated simian immunodeficiency virus (SIVmacC8) confers potent, reproducible protection against homologous wild-type virus challenge (SIVmacJ5). The ability of SIVmacC8 to confer resistance to superinfection with an uncloned ex vivo derivative of SIVmac251 (SIVmac32H/L28) was investigated. In naïve, Mauritian-derived cynomolgus macaques (Macaca fascicularis), SIVmac32H/L28 replicated to high peak titres (>10(8) SIV RNA copies ml(-1)), persisted at high levels and induced distinctive pathology in lymphoid tissues. In cynomolgus macaques vaccinated with SIVmacC8, no evidence of detectable superinfection was observed in 3/8 vaccinates following challenge 3 or 20 weeks later with SIVmac32H/L28. Analyses after SIVmac32H/L28 challenge revealed a significant reduction in viral RNA (P<0.001) and DNA levels between 20 week vaccinates and challenge controls. Amongst 3 week vaccinates, less potent protection was observed. However, analysis of env from breakthrough virus indicated >99% sequence similarity with the vaccine virus. Highly sensitive PCR assays that distinguish vaccine and challenge virus stocks demonstrated restimulation of replication of the vaccine virus SIVmacC8 in the face of potent protection against a vigorous, homologous challenge virus. Vaccine-induced antiviral neutralizing antibodies and anti-Nef CD8+ cytotoxic T cell responses did not correlate with the outcome of the challenge. Defining the mechanism of vaccine protection will need to account for the effective control of a genetically closely related challenge virus whilst remaining unable to suppress replication of the pre-existing vaccine virus. The role of innate and intrinsic anti-retroviral immunity in the protection conferred by live attenuated SIV vaccines warrants careful study.

In order to test the hypothesis that CD8+ cytotoxic T lymphocytes mediate protection against acute superinfection, we depleted >99% of CD8+ lymphocytes in live attenuated simian immunodeficiency virus macC8 (SIVmacC8) vaccinees from the onset of vaccination, maintained that depletion for 20 days, and then challenged with pathogenic, wild-type SIVmacJ5. Vaccinees received 5 mg per kg of humanized anti-CD8 monoclonal antibody (MAb) 1 h before inoculation, followed by the same dose again on days 3, 7, 10, 13, and 17. On day 13, peripheral CD8+ T lymphocytes were >99% depleted in three out of four anti-CD8 MAb-treated vaccinees. At this time attenuated SIVmacC8 viral RNA loads in anti-CD8 MAb-treated vaccinees were significantly higher than control vaccinees treated contemporaneously with nonspecific human immunoglobulin. Lymphoid tissue CD8+ T lymphocyte depletion was >99% in three out of four anti-CD8 MAb-treated vaccinees on the day of wild-type SIVmacJ5 challenge. All four control vaccinees and three out of four anti-CD8 MAb-treated vaccinees were protected against detectable superinfection with wild-type SIVmacJ5. Although superinfection with wild-type SIVmacJ5 was detected at postmortem in a single anti-CD8 MAb-treated vaccinee, this did not correlate with the degree of preceding CD8+ T lymphocyte depletion. Clearance of attenuated SIVmacC8 viremia coincided with recovery of normal CD8+ T lymphocyte counts between days 48 and 76. These results support the view that cytotoxic T lymphocytes are important for host-mediated control of SIV primary viremia but do not indicate a central role in protection against acute superinfection conferred by inoculation with live attenuated SIV.

The efficacy of immunizing with a combination of simian immunodeficiency virus (SIV) Nef vaccines was evaluated. Four vaccinates received three intradermal immunizations with recombinant vaccinia virus that expressed SIV Nef, followed by three intramuscular immunizations with rDNA also expressing SIV Nef. Finally, the four vaccinates received two subcutaneous boosts with recombinant SIV Nef protein. This immunization protocol elicited anti-Nef antibodies in all of the vaccinates as well as specific proliferative responses. However, specific cytotoxic T cell responses were not detected before virus challenge. All vaccinates were challenged intravenously with 10 MID(50) of SIVmacJ5 along with four controls. All eight subjects became infected after SIV challenge and there were no group-specific differences in virus load as measured by virus titration and vRNA analysis. The results of this study support indirectly the report from Gallimore and colleagues (Nat Med 1995;1:1667) suggesting that CD8(+) T lymphocyte responses are required for Nef-based vaccines to restrict SIV infection. If Nef-based vaccines are to be beneficial in controlling infection with immunodeficiency viruses, then it will be necessary to develop more effective immunization protocols that elicit potent CD8(+) cell responses reproducibly.

The experimental infection of macaques with simian immunodeficiency virus (SIV) has provided strong evidence that it may be possible to develop a vaccine against AIDS. Live attenuated SIV vaccines have been found to confer the most potent protection against challenge with a variety of pathogenic viruses. This article summarizes the work performed at NIBSC to characterize the protection conferred by live attenuated SIV and to identify mechanisms of vaccine protection. The results of these experiments are discussed in conjunction with observations from related studies made by other groups.

To determine the role that cellular immune responses play in the protection conferred by vaccination with attenuated SIVmac32H (pC8), we have attempted to deplete macaques of their CD8+ cells prior to challenge with wild-type SIVmac32H (pJ5). In two of four pC8-infected macaques, N109 and N112, a transient partial depletion of CD8+ cells by antibody treatment was achieved. On the day of challenge peripheral CD2+CD4-CD8+ cell counts were reduced by 92 and 95%, respectively, in animals N109 and N112 and their lymph nodes revealed a 46 and 58% reduction, respectively, in CD2+CD4-CD8+ cells. Two other pC8-immunized macaques, N110 and N111, treated in the same way, did not show significant depletion of CD8+ cells. None of these four pC8-immunized animals became infected when challenged with 50 MID50 of pJ5. Treatment of a further four pC8-infected and protected macaques and two naive control animals with Campath-1H antibody successfully depleted peripheral CD3+ cell counts by >99% in all treated animals. Campath-1H depletion resulted in enhanced, longer lasting lymphoid depletion. Yet subsequent challenge with 20 MID50 of pJ5 still failed to infect the pC8-immunized animals. All eight of the naive controls, including two Campath-1H-treated animals, became infected following challenge. In summary, partial depletion of circulating CD8+ cells or total lymphocytes prior to challenge failed to abrogate the protection conferred by vaccination with pC8.

A vaccine against AIDS will probably have to protect against challenge both by viable virus-infected cells and by cell-free virus. Eight cynomolgus macaques infected with attenuated simian immunodeficiency virus (SIV) were challenged (four each) with cell-free and cell-associated SIV. All were protected, whereas eight controls were all infected after challenge. These findings show that live-attenuated vaccine can confer protection against SIV in macaques. Extrapolation to human beings will require extensive evaluation of the safety of attenuated retroviruses. Alternatively, the mechanism of this potent protection must be understood and reproduced by less hazardous means.