Rob Dubbes

Systemic neutralizing antibodies induced by long interval mucosally primed systemically boosted immunization correlate with protection from mucosal SHIV challenge

Immune correlates of vaccine protection from HIV-1 infection would provide important milestones to guide HIV-1 vaccine development. In a proof of concept study using mucosal priming and systemic boosting, the titer of neutralizing antibodies in sera was found to correlate with protection of mucosally exposed rhesus macaques from SHIV infection. Mucosal priming consisted of two sequential immunizations at 12-week intervals with replicating host range mutants of adenovirus type 5 (Ad5hr) expressing the HIV-1(89.6p) env gene. Following boosting with either heterologous recombinant protein or alphavirus replicons at 12-week intervals animals were intrarectally exposed to infectious doses of the CCR5 tropic SHIV(SF162p4). Heterologous mucosal prime systemic boost immunization elicited neutralizing antibodies (Nabs), antibody-dependent cytotoxicity (ADCC), and specific patterns of antibody binding to envelope peptides. Vaccine induced protection did not correlate with the type of boost nor T-cell responses, but rather with the Nab titer prior to exposure.

Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic

Each year, approximately five million people die worldwide from putatively vaccine-preventable mucosally transmitted diseases. With respect to mass vaccination campaigns, one strategy to cope with this formidable challenge is aerosol vaccine delivery, which offers potential safety, logistical, and cost-saving advantages over traditional vaccination routes. Additionally, aerosol vaccination may elicit pivotal mucosal immune responses that could contain or eliminate mucosally transmitted pathogens in a preventative or therapeutic vaccine context. In this current preclinical non-human primate investigation, we demonstrate the feasibility of aerosol vaccination with the recombinant poxvirus-based vaccine vectors NYVAC and MVA. Real-time in vivo scintigraphy experiments with radiolabeled, aerosol-administered NYVAC-C (Clade C, HIV-1 vaccine) and MVA-HPV vaccines revealed consistent mucosal delivery to the respiratory tract. Furthermore, aerosol delivery of the vaccines was safe, inducing no vaccine-associated pathology, in particular in the brain and lungs, and was immunogenic. Administration of a DNA-C/NYVAC-C prime/boost regime resulted in both systemic and anal-genital HIV-specific immune responses that were still detectable 5 months after immunization. Thus, aerosol vaccination with NYVAC and MVA vectored vaccines constitutes a tool for large-scale vaccine efforts against mucosally transmitted pathogens.

Differences in early virus loads with different phenotypic variants of HIV-1 and SIV(cpz) in chimpanzees.

ObjectiveA comparative study of the replication kinetics of different HIV-1 variants (including SIVcpz) was undertaken to determine which viral characteristics were associated with sustained plasma viraemia in chimpanzees. DesignPlasma samples from chimpanzees infected with six different HIV-1 clade B isolates were compared with plasma samples from SIVcpz−ant-infected chimpanzees. MethodsA pan-clade quantitative competitive reverse transcriptase–polymerase chain reaction assay was developed based on conserved primer sequences recognizing M, N and O human lentiviruses as well as different SIVcpz isolates. ResultsImportant differences between early kinetics in the human lentivirus isolates as well as compared with the chimpanzee isolate SIVcpz−ant were observed. R5-dependent non-syncytium-inducing (NSI) isolates (5016, Ba-L, SIVcpz) were found to have relatively higher viral loads than the syncytium-inducing (SI), X4-dependent primary (SF2), T cell-adapted (IIIB) or X4/R5 (Han2, DH12) SI primary isolates. ConclusionInfection of chimpanzees with NSI R5-utilizing isolates correlated with persistent viraemia (approximately 104 RNA equivalents/ml) in contrast to transient viraemia observed after infection with SI X4-utilizing isolates.

Immune strategies utilized by lentivirus infected chimpanzees to resist progression to AIDS.

HIV-1 infected chimpanzees are relatively resistant to the development of AIDS despite their close genetic relatedness to humans and their susceptibility to HIV-1 infection. We have systematically studied possible reasons for their relative ability to maintain T helper (Th) cell numbers and immune competence in the presence of chronic HIV-1 infection. Factors which may alone or together cause the loss in T-cell dependent immunity include: (i) the loss of Th cell function; (ii) the loss of Th cells; and (iii) the loss of capacity for Th cell renewal. Differences in the in vivo and in vitro responses of T lymphocytes from chimpanzees and humans were compared for evidence of HIV-1 related T-cell dysfunction. In contrast to HIV infected individuals, HIV-1 infected chimpanzees maintained strong Th cell proliferative and cytokine responses after receiving tetanus toxoid boosts. In addition there was no abnormal Th1 to Th2 shift as is suggested to occur in AIDS patients. There was no evidence of Th cell dysfunction such as increased level of programmed cell death (PCD) or immune activation in HIV-1 infected chimpanzees in contrast to HIV-1 infected asymptomatic humans. Anergy could be induced with HIV-1 gp120 in human but not chimpanzee Th lymphocytes. We then asked if there was a direct loss of chimpanzee CD4+ cells due to HIV-1 infection in vitro. Infection of chimpanzee CD4+ lymphocyte cultures with HIV-1 in the absence of CD8+ cells resulted in marked cytopathic effect with complete lysis and loss of cells within 3 weeks. We concluded that most chronic HIV-1 infected chimpanzees were able to maintain relatively stable CD4+ lymphocyte numbers despite CD4+ lymphocyte destruction due to direct effects of the virus. Furthermore, there was no evidence of indirect Th cell loss, since neither increased levels of anergy nor apoptosis were observed. Lymph node biopsies from HIV-1 infected chimpanzees revealed that MHC class II rich regions of lymph nodes remained intact, in contrast to the involution of these regions in infected humans. This suggested that chimpanzees may maintain the capacity for Th cell renewal by preserving this MHC class II lymphoid environment. The data presented in this paper suggests that chimpanzees may preserve this critical MHC class II-Th cell environment by dramatically suppressing extra-cellular virus load and that this may be in part mediated by soluble lentivirus suppressing factors.

Virus load in chimpanzees infected with human immunodeficiency virus type 1: effect of pre-exposure vaccination.

Many reports indicate that a long-term asymptomatic state following human immunodeficiency virus type 1 (HIV-1) infection is associated with a low amount of circulating virus. To evaluate the possible effect of stabilizing a low virus load by non-sterilizing pre-exposure vaccination, a quantitative virus isolation method was developed and evaluated in four chronically infected chimpanzees infected with a variety of HIV-1 related isolates. This assay was then used to monitor a group of chimpanzees (n = 6) challenged with HIV-1 following vaccination with gp120 or gp160. Data indicated that of the three vaccinated animals which became infected after challenge, the animal with the lowest neutralizing titre at the time of challenge acquired a virus load similar to the control animals, whereas the two other chimpanzees had reduced numbers of virus producing cells in their peripheral circulation. One animal became virus isolation negative, developed an indeterminant PCR signal on lymph node DNA and subsequently became negative for HIV-1 DNA as determined by PCR on PBMC (peripheral blood mononuclear cells) and bone marrow DNA. Recently, the second animal has also become PCR negative. To confirm observations from quantitative virus isolations, quantification of HIV-1 DNA in PBMC and virus RNA in serum was performed by PCR on serially diluted samples at two different time points. Comparison of virus load as determined by these three methods confirmed that there was an effect of vaccination in reducing virus load and demonstrated a correlation between decreased numbers of virus producing cells, HIV-1 DNA containing cells and virus RNA molecules in serum.

Protection of rhesus macaques from SIV infection by immunization with different experimental SIV vaccines.

The immunogenicity and efficacy of an inactivated whole SIVmac (32H) preparation adjuvanted with muramyl dipeptide (SIV-MDP) and a gp120-enriched SIVmac (32H) ISCOM preparation (SIV-ISCOM), were compared by immunizing four rhesus macaques (Macaca mulatta) four times with SIV-MDP and four others in the same way with SIV-ISCOM. Two monkeys immunized with whole inactivated measles virus (MV) adjuvanted with MDP (MV-MDP) and two monkeys immunized with MV-ISCOM served as controls. In the SIV-ISCOM-immunized monkeys higher SIV-specific serum antibody titres were found than in the SIV-MDP-immunized monkeys. In contrast to the MV-immunized monkeys all SIV-MDP- and SIV-ISCOM-immunized monkeys were protected against intravenous challenge 2 weeks after the last immunization with 10 median monkey infectious doses (MID50) of a cell-free SIVmac (32H) challenge stock propagated in the human T-cell line C8166. After 43 weeks the protected monkeys were reboosted and 2 weeks later rechallenged with 10 MID50 of the same virus produced in peripheral blood mononuclear cells (PBMC) from a rhesus macaque. None of these animals proved to be protected against this challenge. In a parallel experiment in which the same numbers of monkeys were immunized in the same way, the animals were challenged intravenously with 10 MID50 of PBMC from an SIVmac (32H)-infected rhesus macaque. Two out of four SIV-MDP- and two out of four SIV-ISCOM-immunized monkeys proved to be protected from SIV infection.

Molecular Evolution Analysis of the Human Immunodeficiency Virus Type 1 Envelope in Simian/Human Immunodeficiency Virus-Infected Macaques: Implications for Challenge Dose Selection

ABSTRACT Since the demonstration that almost 80% of human immunodeficiency virus type 1 (HIV-1) infections result from the transmission of a single variant from the donor, biological features similar to those of HIV mucosal transmission have been reported for macaques inoculated with simian immunodeficiency virus (SIV). Here we describe the early diversification events and the impact of challenge doses on viral kinetics and on the number of variants transmitted in macaques infected with the chimeric simian/human immunodeficiency virus SHIVsf162p4. We show that there is a correlation between the dose administered and the number of variants transmitted and that certain inoculum variants are preferentially transmitted. This could provide insight into the viral determinants of transmission and could aid in vaccine development. Challenge through the mucosal route with high doses results in the transmission of multiple variants in all the animals. Such an unrealistic scenario could underestimate potential intervention measures. We thus propose the use of molecular evolution analysis to aid in the determination of challenge doses that better mimic the transmission dynamics seen in natural HIV-1 infection.

The rate of progression to AIDS is independent of virus dose in simian immunodeficiency virus-infected macaques.

Of the viral factors that are proposed to influence the rate of progression to AIDS, the role of infectious dose remains unresolved. Intravenous infection of outbred Macaca mulatta with various doses of simian immunodeficiency virus isolate 8980 (SIV(8980)) revealed an endpoint from which an infectious dose 50 (ID(50)) was defined. In the six infected animals, the time to develop AIDS was variable with a spectrum of rapid, intermediate and slow progressors. High and sustained plasma viraemia with marked loss of CD4(+) T-cells was a distinguishing feature between rapid versus intermediate and slow progressors. Animals that received the highest doses did not develop the highest sustained viral loads, nor did they progress more rapidly to disease. Similarly, animals infected with lower doses did not uniformly develop lower viral loads or progress more slowly to AIDS. Furthermore, compiled data from more than 21 animals infected with different doses of the same virus administered by the same route failed to reveal any correlation of infectious dose with survival. Indeed, host factors of these outbred animals, rather than dose of the initial inoculum, were probably an important factor influencing the rate of disease progression in each individual animal. Comparison of animals infected with SIV(B670), from which SIV(8980) was derived, revealed marked differences in disease progression. Clearly, although dose did not influence viral loads nor disease progression, the virulence of the initial inoculum was a major determinant of the rate of progression to AIDS.

CD8+ NK cells are predominant in chimpanzees, characterized by high NCR expression and cytokine production, and preserved in chronic HIV-1 infection

HIV‐1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an “anergic” CD56− CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56bright and CD56dim subset but, as shown here, could be subdivided into functionally different CD8+ and CD8− subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN‐γ, TNF‐α and CD107 than their CD8− counterparts. In addition, chimpanzee CD8− NK cells had relatively high levels of HLA‐DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up‐regulated in CD8+ but not in CD8− NK cells and were functionally capable of inhibiting NKp30‐triggered killing. In contrast to HIV‐1‐infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.

A strategy for cloning infectious molecular clones of retroviruses from serum or plasma

To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.