Expression of Human CD4 and chemokine receptors in cotton rat cells confers permissiveness for productive HIV infection

<p>Abstract</p> <p>Background</p> <p>Current small animal models for studying HIV-1 infection are very limited, and this continues to be a major obstacle for studying HIV-1 infection and pathogenesis, as well as for the urgent development and evaluation of effective anti-HIV-1 therapies and vaccines. Previously, it was shown that HIV-1 can infect cotton rats as indicated by development of antibodies against all major proteins of the virus, the detection of viral cDNA in spleen and brain of challenged animals, the transmission of infectious virus, albeit with low efficiency, from animal to animal by blood, and an additional increase in the mortality in the infected groups.</p> <p>Results</p> <p>Using <it>in vitro </it>experiments, we now show that cotton rat cell lines engineered to express human receptor complexes for HIV-1 (hCD4 along with hCXCR4 or hCCR5) support virus entry, viral cDNA integration, and the production of infectious virus.</p> <p>Conclusion</p> <p>These results further suggest that the development of transgenic cotton rats expressing human HIV-1 receptors may prove to be useful small animal model for HIV infection.</p

Comparative Expression of Renin-Angiotensin Pathway Proteins in Visceral Versus Subcutaneous Fat

Body fat distribution contributes to obesity-related metabolic and cardiovascular disorders. Visceral fat is more detrimental than subcutaneous fat. However, the mechanisms underlying visceral fat-mediated cardiometabolic dysregulation are not completely understood. Localized increases in expression of the renin angiotensin system (RAS) in adipose tissue (AT) may be implicated. We therefore investigated mRNA and protein expression of RAS components in visceral versus subcutaneous AT using paired samples from individuals undergoing surgery (N = 20, body mass index: 45.6 ± 6.2 kg/m2, and age: 44.6 ± 9.1 years). We also examined RAS-related proteins in AT obtained from individuals on renin angiotensin aldosterone system (RAAS) targeted drugs (N = 10, body mass index: 47.2 ± 9.3 kg/m2, and age: 53.3 ± 10.1 years). Comparison of protein expression between subcutaneous and visceral AT samples showed an increase in renin (p = 0.004) and no change in angiotensinogen (p = 0.987) expression in visceral AT. Among proteins involved in angiotensin peptide generation, angiotensin converting enzyme (p = 0.02) was increased in subcutaneous AT while chymase (p = 0.001) and angiotensin converting enzyme-2 (p = 0.001) were elevated in visceral fat. Furthermore, visceral fat expression of angiotensin II type-2 receptor (p = 0.007) and angiotensin II type-1 receptor (p = 0.031) was higher, and MAS receptor (p &lt; 0.001) was lower. Phosphorylated-p53 (p = 0.147), AT fibrosis (p = 0.138) and average adipocyte size (p = 0.846) were similar in the two depots. Nonetheless, visceral AT showed increased mRNA expression of inflammatory (TNFα, p &lt; 0.001; IL-6, p = 0.001) and oxidative stress markers (NOX2, p = 0.038; NOX4, p &lt; 0.001). Of note, mRNA and protein expression of RAS components did not differ between subjects taking or not taking RAAS related drugs. In summary, several RAS related proteins are differentially expressed in subcutaneous versus visceral AT. This differential expression may not alter AngII but likely increases Ang1-7 generation in visceral fat. These potential differences in active angiotensin peptides and receptor expression in the two depots suggest that localized RAS may not be involved in differences in visceral vs subcutaneous AT function in obese individuals. Our findings do not support a role for localized RAS differences in visceral fat-mediated development of cardiovascular and metabolic pathology

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report II

BACKGROUND: Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). METHODS: In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. FINDINGS: Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. CONCLUSIONS: Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation

Phase I Safety and Immunogenicity Evaluation of MVA-CMDR, a Multigenic, Recombinant Modified Vaccinia Ankara-HIV-1 Vaccine Candidate

We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes env/gag/pol. The HIV sequences were derived from circulating recombinant form CRF01_AE, which predominates in Thailand. The objective was to evaluate safety and immunogenicity of MVA-CMDR in human volunteers in the US and Thailand.MVA-CMDR or placebo was administered intra-muscularly (IM; 10(7) or 10(8) pfu) or intradermally (ID; 10(6) or 10(7) pfu) at months 0, 1 and 3, to 48 healthy volunteers at low risk for HIV-1 infection. Twelve volunteers in each dosage group were randomized to receive MVA-CMDR or placebo (10∶2). Volunteers were actively monitored for local and systemic reactogenicity and adverse events post vaccination. Cellular immunogenicity was assessed by a validated IFNγ Elispot assay, an intracellular cytokine staining assay, lymphocyte proliferation and a (51)Cr-release assay. Humoral immunogenicity was assessed by ADCC for gp120 and binding antibody ELISAs for gp120 and p24. MVA-CMDR was safe and well tolerated with no vaccine related serious adverse events. Cell-mediated immune responses were: (i) moderate in magnitude (median IFNγ Elispot of 78 SFC/10(6) PBMC at 10(8) pfu IM), but high in response rate (70% (51)Cr-release positive; 90% Elispot positive; 100% ICS positive, at 10(8) pfu IM); (ii) predominantly HIV Env-specific CD4(+) T cells, with a high proliferative capacity and durable for at least 6 months (100% LPA response rate by the IM route); (iv) dose- and route-dependent with 10(8) pfu IM being the most immunogenic treatment. Binding antibodies against gp120 and p24 were detectable in all vaccination groups with ADCC capacity detectable at the highest dose (40% positive at 10(8) pfu IM).MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and elicited durable cell-mediated and humoral immune responses.ClinicalTrials.gov NCT00376090

The Role of Natural Killer (NK) Cells and NK Cell Receptor Polymorphisms in the Assessment of HIV-1 Neutralization

The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses

Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose

Background: A key goal for HIV-1 envelope immunogen design is the induction of cross-reactive neutralizing antibodies (nAbs). As AIDS vaccine recipients will not be exposed to strains exactly matching any immunogens due to multiple HIV-1 quasispecies circulating in the human population worldwide, heterologous SHIV challenges are essential for realistic vaccine efficacy testing in primates. We assessed whether polyclonal IgG, isolated from rhesus monkeys (RMs) with high-titer nAbs (termed SHIVIG), could protect RMs against the R5-tropic tier-2 SHIV-2873Nip, which was heterologous to the viruses or HIV-1 envelopes that had elicited SHIVIG. Results: SHIVIG demonstrated binding to HIV Gag, Tat, and Env of different clades and competed with the broadly neutralizing antibodies b12, VRC01, 4E10, and 17b. SHIVIG neutralized tier 1 and tier 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased the neutralizing activity of SHIVIG 20-fold in PBMC assays. Although SHIVIG neutralized SHIV-2873Nip in vitro, this polyclonal IgG preparation failed to prevent acquisition after repeated intrarectal low-dose virus challenges, but at a dose of 400 mg/kg, it significantly lowered peak viremia (P = 0.001). Unexpectedly, single-genome analysis revealed a higher number of transmitted variants at the low dose of 25 mg/kg, implying increased acquisition at low SHIVIG levels. In vitro, SHIVIG demonstrated complement-mediated Ab-dependent enhancement of infection (C’-ADE) at concentrations similar to those observed in plasmas of RMs treated with 25 mg/kg of SHIVIG. Conclusion: Our primate model data suggest a dual role for polyclonal anti-HIV-1 Abs depending on plasma levels upon virus encounter