Helena Ong

Molecularly Cloned SHIV-1157ipd3N4: a Highly Replication- Competent, Mucosally Transmissible R5 Simian-Human Immunodeficiency Virus Encoding HIV Clade C env

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three of which developed peripheral CD4+ T-cell depletion. After the first inoculated monkey developed AIDS at week 137 postinoculation, transfer of its infected blood to a naïve animal induced memory T-cell depletion and thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-1157ipd3, an extra NF-κB binding site was engineered into its 3′ long terminal repeat, giving rise to SHIV-1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent, mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting HIV-1 clade C Env.

SHIV-1157i and passaged progeny viruses encoding R5 HIV-1 clade C env cause AIDS in rhesus monkeys

BackgroundInfection of nonhuman primates with simian immunodeficiency virus (SIV) or chimeric simian-human immunodeficiency virus (SHIV) strains is widely used to study lentiviral pathogenesis, antiviral immunity and the efficacy of AIDS vaccine candidates. SHIV challenges allow assessment of anti-HIV-1 envelope responses in primates. As such, SHIVs should mimic natural HIV-1 infection in humans and, to address the pandemic, encode HIV-1 Env components representing major viral subtypes worldwide.ResultsWe have developed a panel of clade C R5-tropic SHIVs based upon env of a Zambian pediatric isolate of HIV-1 clade C, the worlds most prevalent HIV-1 subtype. The parental infectious proviral clone, SHIV-1157i, was rapidly passaged through five rhesus monkeys. After AIDS developed in the first animal at week 123 post-inoculation, infected blood was infused into a sixth monkey. Virus reisolated at this late stage was still exclusively R5 tropic and mucosally transmissible. Here we describe the long-term follow-up of this initial cohort of six monkeys. Two have remained non-progressors, whereas the other four gradually progressed to AIDS within 123–270 weeks post-exposure. Two progressors succumbed to opportunistic infections, including a case of SV40 encephalitis.ConclusionThese data document the disease progression induced by the first mucosally transmissible, pathogenic R5 non-clade B SHIV and suggest that SHIV-1157i-derived viruses, including the late-stage, highly replication-competent SHIV-1157ipd3N4 previously described (Song et al., 2006), display biological characteristics that mirror those of HIV-1 clade C and support their expanded use for AIDS vaccine studies in nonhuman primates.

Acute Schistosoma mansoni Infection Increases Susceptibility to Systemic SHIV Clade C Infection in Rhesus Macaques after Mucosal Virus Exposure

Background Individuals living in sub-Saharan Africa represent 10% of the worlds population but almost 2/3 of all HIV-1/AIDS cases. The disproportionate HIV-1 infection rates in this region may be linked to helminthic parasite infections that affect many individuals in the developing world. However, the hypothesis that parasite infection increases an individuals susceptibility to HIV-1 has never been prospectively tested in a relevant in vivo model. Methodology/Principal Findings We measured whether pre-existing infection of rhesus monkeys with a parasitic worm would facilitate systemic infection after mucosal AIDS virus exposure. Two groups of animals, one consisting of normal monkeys and the other harboring Schistosoma mansoni, were challenged intrarectally with decreasing doses of R5-tropic clade C simian-human immunodeficiency virus (SHIV-C). Systemic infection occurred in parasitized monkeys at viral doses that remained sub-infectious in normal hosts. In fact, the 50% animal infectious (AID50) SHIV-C dose was 17-fold lower in parasitized animals compared to controls (P<0.001). Coinfected animals also had significantly higher peak viral RNA loads than controls (P<0.001), as well as increased viral replication in CD4+ central memory cells (P = 0.03). Conclusions/Significance Our data provide the first direct evidence that acute schistosomiasis significantly increases the risk of de novo AIDS virus acquisition, and the magnitude of the effect suggests that control of helminth infections may be a useful public health intervention to help decrease the spread of HIV-1.

Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody That Recognizes a Novel Conformational Epitope on gp41 and Lacks Reactivity against Self-Antigens

ABSTRACT Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.

schistosoma mansoni infection promotes Shiv clade C replication in rhesus macaques

Objective:To evaluate the hypothesis that parasitic infections that induce T-helper type 2 (Th2) immune responses, such as schistosomiasis, upregulate HIV-1 replication. Design:The effect of concomitant Schistosoma mansoni infection was tested in a primate model of acute and chronic simian-human immunodeficiency virus (SHIV) infection in rhesus macaques using a novel SHIV strain encoding the R5 env gene of a primary HIV clade C isolate from sub-Saharan Africa. Methods:S. mansoni-infected rhesus macaques and controls were exposed to SHIV to assess the effects of schistosomiasis on acute viral infection. Effects on chronic viral infection were evaluated by exposing virus-infected animals to parasites. S. mansoni infection was confirmed by the presence of parasite eggs in stool and eosinophilia. Viral RNA loads, cytokine and chemokine mRNA expression were measured by real time reverse transcription–PCR. Results:S. mansoni coinfection increased the expression of Th2-associated cytokine responses and SHIV replication during both acute and chronic phases of SHIV infection. Conclusions:These results support the hypothesis that concomitant schistosomiasis upregulates replication of immunodeficiency viruses in coinfected hosts, raising the possibility that parasite-infected individuals may also be more susceptible to acquisition of HIV-1 infection.

Neutralization-Sensitive R5-Tropic Simian-Human Immunodeficiency Virus SHIV-2873Nip, Which Carries env Isolated from an Infant with a Recent HIV Clade C Infection

ABSTRACT Human immunodeficiency virus clade C (HIV-C) accounts for >56% of all HIV infections worldwide. To investigate vaccine safety and efficacy in nonhuman primates, a pathogenic, R5-tropic, neutralization-sensitive simian-human immunodeficiency virus (SHIV) carrying HIV-C env would be desirable. We have constructed SHIV-2873Ni, an R5-tropic SHIV carrying a primary pediatric HIV-C env gene isolated from a 2-month-old Zambian infant, who died within 1 year of birth. SHIV-2873Ni was constructed using SHIV-1157ipd3N4 (R. J. Song, A. L. Chenine, R. A. Rasmussen, C. R. Ruprecht, S. Mirshahidi, R. D. Grisson, W. Xu, J. B. Whitney, L. M. Goins, H. Ong, P. L. Li, E. Shai-Kobiler, T. Wang, C. M. McCann, H. Zhang, C. Wood, C. Kankasa, W. E. Secor, H. M. McClure, E. Strobert, J. G. Else, and R. M. Ruprecht. J. Virol. 80:8729-8738, 2006) as the backbone, since the latter contains additional NF-κB sites in the long terminal repeats to enhance viral replicative capacity. The parental virus, SHIV-2873Ni, was serially passaged through five rhesus monkeys (RMs); SHIV-2873Nip, the resulting passaged virus, was reisolated from the fourth recipient about 1 year postinoculation. SHIV-2873Nip was replication competent in RM peripheral blood mononuclear cells of all random donors tested and was exclusively R5 tropic, and its env gene clustered with HIV-C by phylogenetic analysis; its high sensitivity to neutralization led to classification as a tier 1 virus. Indian-origin RMs were inoculated by different mucosal routes, resulting in high peak viral RNA loads. Signs of virus-induced disease include depletion of gut CD4+ T lymphocytes, loss of memory T cells in blood, and thrombocytopenia that resulted in fatal cerebral hemorrhage. SHIV-2873Nip is a highly replication-competent, mucosally transmissible, pathogenic R5-tropic virus that will be useful to study viral pathogenesis and to assess the efficacy of immunogens targeting HIV-C Env.

Coinfection with Schistosoma mansoni reactivates viremia in rhesus macaques with chronic simian-human immunodeficiency virus clade C infection.

ABSTRACT We tested the hypothesis that helminth parasite coinfection would intensify viremia and accelerate disease progression in monkeys chronically infected with an R5 simian-human immunodeficiency virus (SHIV) encoding a human immunodeficiency virus type 1 (HIV-1) clade C envelope. Fifteen rhesus monkeys with stable SHIV-1157ip infection were enrolled into a prospective, randomized trial. These seropositive animals had undetectable viral RNA and no signs of immunodeficiency. Seven animals served as virus-only controls; eight animals were exposed to Schistosoma mansoni cercariae. From week 5 after parasite exposure onward, coinfected animals shed eggs in their feces, developed eosinophilia, and had significantly higher mRNA expression of the T-helper type 2 cytokine interleukin-4 (P = 0.001) than animals without schistosomiasis. Compared to virus-only controls, viral replication was significantly increased in coinfected monkeys (P = 0.012), and the percentage of their CD4+ CD29+ memory cells decreased over time (P = 0.05). Thus, S. mansoni coinfection significantly increased viral replication and induced T-cell subset alterations in monkeys with chronic SHIV clade C infection.

Inducing cross-clade neutralizing antibodies against HIV-1 by immunofocusing.

Background Although vaccines are important in preventing viral infections by inducing neutralizing antibodies (nAbs), HIV-1 has proven to be a difficult target and escapes humoral immunity through various mechanisms. We sought to test whether HIV-1 Env mimics may serve as immunogens. Methodology/Principal Findings Using random peptide phage display libraries, we identified the epitopes recognized by polyclonal antibodies of a rhesus monkey that had developed high-titer, broadly reactive nAbs after infection with a simian-human immunodeficiency virus (SHIV) encoding env of a recently transmitted HIV-1 clade C (HIV-C). Phage peptide inserts were analyzed for conformational and linear homology using computational analysis; some peptides mimicked various domains of the original HIV-C Env, such as conformational V3 loop epitopes and the conserved linear region of the gp120 C-terminus. Next, we devised a novel prime/boost strategy to test the immunogenicity of such phage-displayed peptides and primed mice only once with HIV-C gp160 DNA followed by boosting with mixtures of recombinant phages. Conclusions/Significance This strategy, which was designed to focus the immune system on a few Env epitopes (immunofocusing), not only induced HIV-C gp160 binding antibodies and cross-clade nAbs, but also linked a conserved HIV Env region for the first time to the induction of nAbs: the C-terminus of gp120. The identification of conserved antigen mimics may lead to novel immunogens capable of inducing broadly reactive nAbs.

Efficacy of a multigenic protein vaccine containing multimeric HIV gp160 against heterologous SHIV clade C challenges.

Objective:To determine whether multigenic protein immunogens including native, trimeric HIV clade C (HIV-C) gp160 could cross-protect macaques against mucosal challenge with clade C (SHIV-C) mismatched for env. Design:Because AIDS vaccine recipients are unlikely to encounter exactly matched HIV strains and to represent the diversity of locally circulating HIV-C strains, we selected env genes to generate the gp160 immunogen and SHIV-C from different, recently infected infants of the same clinical cohort in Zambia. In a model of postnatal HIV-C transmission, infant macaques were immunized with soluble viral proteins, including trimeric HIV1084i Env, and challenged with SHIV-1157ip; protein-only vaccination was compared with a DNA prime/protein boost strategy. Methods:All vaccinated and control monkeys were exposed orally to low-dose, R5-tropic SHIV-1157ip encoding heterologous env. Animals with no or only transient infection were rechallenged intrarectally with a high dose of R5 SHIV-1157ipd3N4, a ‘late’, animal-evolved SHIV-1157ip variant. Animals were followed prospectively for immune parameters and viral RNA loads. Results:Vaccination induced cross-neutralizing antibodies. Compared to controls, vaccinees had significantly lower peak viral RNA loads, and one vaccine recipient remained completely virus-free, even in lymphoid tissues. There was a trend for the protein-only vaccine to yield better protection than the combined modality approach. Conclusion:Protein-only immunogens induced significant protection against heterologous viruses encoding env from locally circulating viruses.