Jonathan L. Heeney

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

Background The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. Methods and Findings We immortalized IgG+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. Conclusions This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses

The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon γ enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/106 mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen.

Viruses as vaccine vectors for infectious diseases and cancer

Recent developments in the use of viruses as vaccine vectors have been facilitated by a better understanding of viral biology. Advances occur as we gain greater insight into the interrelationship of viruses and the immune system. Viral-vector vaccines remain the best means to induce cellular immunity and are now showing promise for the induction of strong humoral responses. The potential benefits for global health that are offered by this field reflect the scope and utility of viruses as vaccine vectors for human and veterinary applications, with targets ranging from certain types of cancer to a vast array of infectious diseases.

Independent evolution of an antiviral TRIMCyp in rhesus macaques

The antiretroviral restriction factor TRIM5 has recently emerged as an important mediator of innate immunity and species-specific inhibition of retroviral replication in mammals. Selection pressure from pathogenic infection has driven rapid evolution of TRIM5 genes, leading to the antiviral specificities we see today. Remarkably, the New World owl monkey (Aotus trivirgatus) encodes a TRIM5 protein in which the antiviral determinants in the B30.2 domain have been replaced by cyclophilin A (CypA) encoded by a retrotransposed cDNA. The owl monkey TRIMCyp protein restricts infection by a subset of lentiviruses that recruit CypA to their capsids, including HIV-1 and feline immunodeficiency virus. Here, we show that the Old World monkey, rhesus macaque (Macaca mulatta), also encodes a TRIMCyp protein that has arisen independently from that in owl monkeys. The rhesus TRIMCyp is encoded by a single, but common, allele (Mamu7) of the rhesus TRIM5 gene, among at least six further alleles that encode full-length TRIM5 proteins with no homology to CypA. The antiviral specificity of the rhesus TRIMCyp is distinct, restricting infection of HIV-2 and feline immunodeficiency virus but not HIV-1. Restriction by rhesus TRIMCyp is before reverse transcription and inhibited by blocking CypA binding, with cyclosporine A, or by mutation of the capsid CypA binding site. These observations suggest a mechanism of restriction that is conserved between TRIMCyp proteins. The lack of activity against HIV-1 suggests that Mamu7 homozygous animals will be null for TRIM5-mediated restriction of HIV-1 and could contribute to improved animal models for HIV/AIDS.

Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.

Prevention of SIV rectal transmission and priming of T cell responses in macaques after local pre-exposure application of tenofovir gel.

Background The rectum is particularly vulnerable to HIV transmission having only a single protective layer of columnar epithelium overlying tissue rich in activated lymphoid cells; thus, unprotected anal intercourse in both women and men carries a higher risk of infection than other sexual routes. In the absence of effective prophylactic vaccines, increasing attention is being given to the use of microbicides and preventative antiretroviral (ARV) drugs. To prevent mucosal transmission of HIV, a microbicide/ARV should ideally act locally at and near the virus portal of entry. As part of an integrated rectal microbicide development programme, we have evaluated rectal application of the nucleotide reverse transcriptase (RT) inhibitor tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy) propyl] adenine monohydrate), a drug licensed for therapeutic use, for protective efficacy against rectal challenge with simian immunodeficiency virus (SIV) in a well-established and standardised macaque model. Methods and Findings A total of 20 purpose-bred Indian rhesus macaques were used to evaluate the protective efficacy of topical tenofovir. Nine animals received 1% tenofovir gel per rectum up to 2 h prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given tenofovir gel 2 h after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a noncloned, virulent stock of SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from peripheral blood mononuclear cells (PBMC), quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive quantitative competitive (qc) RT-PCR, and presence of SIV-specific serum antibodies by ELISA. We report here a significant protective effect (p = 0.003; Fisher exact probability test) wherein eight of nine macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving tenofovir gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. We found a strong positive association between the concentration of tenofovir in the plasma 15 min after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged tenofovir-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of tenofovir. Intriguingly, in the absence of seroconversion, Gag-specific gamma interferon (IFN-γ)-secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/106 PBMC to 261 spot forming cells (SFC)/106 PBMC. Conclusions These results indicate that colorectal pretreatment with ARV drugs, such as tenofovir, has potential as a clinically relevant strategy for the prevention of HIV transmission. We conclude that plasma tenofovir concentration measured 15 min after rectal administration may serve as a surrogate indicator of protective efficacy. This may prove to be useful in the design of clinical studies. Furthermore, in vitro intestinal explants served as a model for drug distribution in vivo and susceptibility to virus infection. The finding of T cell priming following exposure to virus in the absence of overt infection is provocative. Further studies would reveal if a combined modality microbicide and vaccination strategy is feasible by determining the full extent of local immune responses induced and their protective potential.

Evidence for an ancient selective sweep in the MHC class I gene repertoire of chimpanzees

MHC class I molecules play an essential role in the immune defense against intracellular infections. The hallmark of the MHC is its extensive degree of polymorphism at the population level. However, the present comparison of MHC class I gene intron variation revealed that chimpanzees have experienced a severe repertoire reduction at the orthologues of the HLA-A, -B, and -C loci. The loss of variability predates the (sub)speciation of chimpanzees and did not effect other known gene systems. Therefore the selective sweep in the MHC class I gene may have resulted from a widespread viral infection. Based on the present results and the fact that chimpanzees have a natural resistance to the development of AIDS, we hypothesize that the selective sweep was caused by the chimpanzee-derived simian immunodeficiency virus (SIVcpz), the closest relative of HIV-1, or a closely related retrovirus. Hence, the contemporary chimpanzee populations represent the offspring of AIDS-resistant animals, the survivors of a HIV-like pandemic that took place in the distant past.

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

ABSTRACT Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-γ), protein-specific lymphoproliferative responses, IFN-γ, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

HIV-1 envelope-elicited neutralizing antibody titres correlate with protection and virus load in chimpanzees

In an attempt to compare the protective effect of vaccination with two forms of envelope antigens, and to define immunological correlates of protection against HIV infection, chimpanzees were vaccinated with either recombinant gp160 or gp120. Homologous HIV challenge was performed 3 weeks after the fourth immunization. The animal with the highest level of serum neutralizing antibodies (gp160 immunogen) was protected against HIV infection. All other chimpanzees became infected, but displayed various levels of infected PBMCs. The postchallenge data gave rise to the following conclusions: (1) protection correlated with the level of the serological immune response, but not with the nature of immunogen (gp120 versus gp160); (2) the virus-neutralizing titre at day of challenge correlated with protection from infection; (3) the relative magnitude of the lymphoproliferative T-cell response at day of challenge did not correlate with any protective effect; (4) the peak numbers of virus-infected PBMCs in vaccinated animals were lower than those observed in control animals, and this effect was correlated with the intensity of the antibody response at day of challenge. This raises the possibility that a beneficial effect of HIV vaccination may be achieved in a situation where sterile immunity is not consistently obtained.