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The main advantage of animal models of infectious diseases over in vitro studies is the gain in the understanding of the complex dynamics between the immune system and the pathogen. While small animal models have practical advantages over large animal models, it is crucial to be aware of their limitations. Although the small animal model at least needs to be susceptible to the pathogen under study to obtain meaningful data, key elements of pathogenesis should also be reflected when compared to humans. Well-designed small animal models for HIV, hepatitis viruses and tuberculosis require, additionally, a thorough understanding of the similarities and differences in the immune responses between humans and small animals and should incorporate that knowledge into the goals of the study. To discuss these considerations, the NIAID hosted a workshop on ‘Small Animal Models for HIV, Hepatitis B, and Tuberculosis’ on May 30, 2019. Highlights of the workshop are outlined below.

Over 30 key leaders in the field participated in a 1-day workshop entitled ‘Recent Advances and Opportunities in the Development and Use of Humanized Immune System Mouse Models’ to discuss the benefits and limitations of using human fetal tissue versus non-fetal tissue sources to generate mice with a humanized immune system. This Comment summarizes the workshop discussions, including highlights of some of the key advances made through the use of humanized mice in improving the understanding of immune system function and developing novel therapeutics for the treatment of infectious, immunological and allergic diseases, as well as current challenges in the production, characterization and utilization of these animal models.

Mouse monoclonal antibodies with varying specificities against the Gag capsid of simian and human immunodeficiency virus (SIV/HIV) were generated by immunizing mice with whole inactivated SIVagmTYO-1. Monoclonal antibody AG3.0 showed the broadest reactivity recognizing the Gag capsid protein (p24–27) and Gag precursors p38, p55, and p150 of HIV-1, HIV-2, SIVmac, and SIVagm. Using overlapping peptides, the AG3.0 epitope was mapped in capsid to a sequence (SPRTLNA) conserved among HIV-1, HIV-2, SIVrcm, SIVsm/mac, and SIVagm related viruses. Because of its broad cross-reactivity, AG3.0 was used to develop an antigen capture assay with a lower detection limit of 100pg/ml HIV-1 Gag p24. Interestingly, AG3.0 was found to have a faster binding on/off rate for SIVagmVer and SIVmac Gag than for SIVagmSab Gag, possibly due to differences outside the SPRTLNA motif. In addition, the ribonucleic acid (RNA) coding for AG3.0 was sequenced to facilitate the development of humanized monoclonal antibodies.

Most human immunodeficiency virus (HIV) transmissions in women occur through the cervicovaginal mucosa, which is coated by a bacterial biofilm including Lactobacillus. This commensal bacterium has a role in maintaining a healthy mucosa and can be genetically engineered to produce antiviral peptides. Here, we report a 63% reduction in transmission of a chimeric simian/HIV (SHIV(SF162P3)) after repeated vaginal challenges of macaques treated with Lactobacillus jensenii expressing the HIV-1 entry inhibitor cyanovirin-N. Furthermore, peak viral loads in colonized macaques with breakthrough infection were reduced sixfold. Colonization and prolonged antiviral protein secretion by the genetically engineered lactobacilli did not cause any increase in proinflammatory markers. These findings lay the foundation for an accessible and durable approach to reduce heterosexual transmission of HIV in women, which is coitally independent, inexpensive, and enhances the natural protective effects of the vaginal microflora.

Many species of African nonhuman primates are natural hosts for individual strains of simian immunodeficiency virus (SIV). These infected animals do not, however, develop AIDS. Here we show that multiple species of African nonhuman primate species characteristically have low frequencies of CD4 + T cells and high frequencies of both T cells that express only the alpha-chain of CD8 and double-negative T cells. These subsets of T cells are capable of eliciting functions generally associated with CD4 + T cells, yet these cells lack surface expression of the CD4 protein and are, therefore, poor targets for SIV in vivo . These data demonstrate that coevolution with SIV has, in several cases, involved downregulation of receptors for the virus by otherwise-susceptible host target cells. Understanding the genetic factors that lead to downregulation of these receptors may lead to therapeutic interventions that mimic this modulation in progressive infections.

An SIV-infected rhesus macaque presented with anemia, hypercalcemia, and hyperglobulinemia. Neoplastic round cells with plasma cell morphology infiltrated multiple organs and stained immunohistochemically positive for CD45, MUM1/IRF4, CD138, VS38C, and Kappa light chain and variably positive for CD20 and CD79a; consistent with a B-cell neoplasm with plasma cell differentiation.

Since the 1980s, the rapid progression of the HIV/AIDS pandemic has prompted a major international research effort. As a result, the current knowledge on HIV biology, its evolution, and origins [1–5] exceeds that of many, if not all, other viruses. One of the most important areas of HIV research is the detailed understanding of HIV replication. As with all viruses, HIV must exploit the host’s cellular machinery and metabolism to copy its genetic material, synthesize viral proteins, and assemble new virions. The viral replication cycle is thus dependent on an intricate network of direct and indirect protein interactions: between the viral proteins, between the virus and the host, and ultimately between the various host proteins that constitute the subverted cellular systems. When we also take into account the host’s immune response and intrinsic antiviral factors, there are clearly a large number of host–pathogen relationships that are important to our full understanding of HIV biology. However, until recently [6], this valuable information has remained ‘locked’ in the published literature, making it time-consuming to study by individual researchers and inaccessible to computational analysis, thus hindering the progress of research. Here we highlight new developments in the area of host–pathogen systems biology that in our opinion will provide helpful insights to the HIVresearch community.

Although many interactions between HIV-1 and human proteins have been reported in the scientific literature, no publicly accessible source for efficiently reviewing this information was available. Therefore, a project was initiated in an attempt to catalogue all published interactions between HIV-1 and human proteins. HIV-related articles in PubMed were used to develop a database containing names, Entrez GeneIDs, and RefSeq protein accession numbers of interacting proteins. Furthermore, brief descriptions of the interactions, PubMed identification numbers of articles describing the interactions, and keywords for searching the interactions were incorporated. Over 100,000 articles were reviewed, resulting in the identification of 1448 human proteins that interact with HIV-1 comprising 2589 unique HIV-1-to-human protein interactions. Preliminary analysis of the extracted data indicates 32% were direct physical interactions (e.g., binding) and 68% were indirect interactions (e.g., upregulation through activation of signaling pathways). Interestingly, 37% of human proteins in the database were found to interact with more than one HIV-1 protein. For example, the signaling protein mitogen-activated protein kinase 1 has a surprising range of interactions with 10 different HIV-1 proteins. Moreover, large numbers of interactions were published for the HIV-1 regulatory protein Tat and envelope proteins: 30% and 33% of total interactions identified, respectively. The database is accessible at http://www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions/ and is cross-linked to other National Center for Biotechnology Information databases and programs via Entrez Gene. This database represents a unique and continuously updated scientific resource for understanding HIV-1 replication and pathogenesis to assist in accelerating the development of effective therapeutic and vaccine interventions.

The immunologic and virologic outcome of therapeutic DNA-vaccines administered during antiretroviral therapy (ART) using electroporation with or without (interleukin) IL-2 treatment was evaluated in the SIV-mac239/macaque model. Rhesus macaques inoculated with pathogenic SIVmac239 were treated with ART [(R(-9-(2-phosphonomethoxypropyl) adenine) (PMPA), FTC, Zerit®] from weeks 13 to 41 postinfection (wpi). Group 1 (n = 7) received ART only, groups 2 and 3 (each n = 6) additionally received SIVmac239-derived gp140Env, GagPol, and TatRevNef plasmids by in vivo electroporation at 22, 26, 30, and 34 wpi, and group 3 also IL-2 for 14 days after each vaccination. Endpoints evaluated were viral load, Gag181–189-specific CD8+ T-cell responses in MamuA01+ animals, lymphoproliferative responses, and CD4 T-cell counts. Viremia in all animals dropped below 200 RNA copies/ml during ART. Frequencies of Gag181–189-specific CD8+ T cells prior to ART were detectable in all three groups (1.27–3.01%) and increased significantly (p

Topical microbicides are self-administered, prophylactic products for protection against sexually transmitted pathogens. A large number of compounds with known anti-human immunodeficiency virus type 1 (HIV-1) inhibitory activity have been proposed as candidate topical microbicides. To identify potential leads, an in vitro screening algorithm was developed to evaluate candidate microbicides in assays that assess inhibition of cell-associated and cell-free HIV-1 transmission, entry, and fusion. The algorithm advances compounds by evaluation in a series of defined assays that generate measurements of relative antiviral potency to determine advancement or failure. Initial testing consists of a dual determination of inhibitory activity in the CD4-dependent CCR5-tropic cell-associated transmission inhibition assay and in the CD4/CCR5-mediated HIV-1 entry assay. The activity is confirmed by repeat testing, and identified actives are advanced to secondary screens to determine their effect on transmission of CXCR4-tropic viruses in the presence or absence of CD4 and their ability to inhibit CXCR4- and CCR5-tropic envelope-mediated cell-to-cell fusion. In addition, confirmed active compounds are also evaluated in the presence of human seminal plasma, in assays incorporating a pH 4 to 7 transition, and for growth inhibition of relevant strains of lactobacilli. Leads may then be advanced for specialized testing, including determinations in human cervical explants and in peripheral blood mononuclear cells against primary HIV subtypes, combination testing with other inhibitors, and additional cytotoxicity assays. PRO 2000 and SPL7013 (the active component of VivaGel), two microbicide products currently being evaluated in human clinical trials, were tested in this in vitro algorithm and were shown to be highly active against CCR5- and CXCR4-tropic HIV-1 infection.

In the absence of an effective vaccine, there is an urgent need for safe and effective antiviral agents to prevent transmission of HIV. Here, we report that an amphipathic α-helical peptide derived from the hepatitis C virus NS5A anchor domain (designated C5A in this article) that has been shown to be virocidal for the hepatitis C virus (HCV) also has potent antiviral activity against HIV. C5A exhibits a broad range of antiviral activity against HIV isolates, and it prevents infection of the three in vivo targets of HIV: CD4 + T lymphocytes, macrophages, and dendritic cells by disrupting the integrity of the viral membrane and capsid core while preserving the integrity of host membranes. C5A can interrupt an ongoing T cell infection, and it can prevent transmigration of HIV through primary genital epithelial cells, infection of mucosal target cells and transfer from dendritic cells to T cells ex vivo , justifying future experiments to determine whether C5A can prevent HIV transmission in vivo .

The number of humanized mouse models for the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and other infectious diseases has expanded rapidly over the past 8 years. Highly immunodeficient mouse strains, such as NOD/SCID/gamma chain(null) (NSG, NOG), support better human hematopoietic cell engraftment. Another improvement is the derivation of highly immunodeficient mice, transgenic with human leukocyte antigens (HLAs) and cytokines that supported development of HLA-restricted human T cells and heightened human myeloid cell engraftment. Humanized mice are also used to study the HIV reservoir using new imaging techniques. Despite these advances, there are still limitations in HIV immune responses and deficits in lymphoid structures in these models in addition to xenogeneic graft-versus-host responses. To understand and disseminate the improvements and limitations of humanized mouse models to the scientific community, the NIH sponsored and convened a meeting on April 15, 2015 to discuss the state of knowledge concerning these questions and best practices for selecting a humanized mouse model for a particular scientific investigation. This report summarizes the findings of the NIH meeting.

Background At present, there is no effective vaccine or other approved product for the prevention of sexually transmitted human immunodeficiency virus type 1 (HIV-1) infection. It has been reported that women in resource-poor communities use vaginally applied citrus juices as topical microbicides. These easily accessible food products have historically been applied to prevent pregnancy and sexually transmitted diseases. The aim of this study was to evaluate the efficacy and cytotoxicity of these substances using an established topical microbicide testing algorithm. Freshly squeezed lemon and lime juice and household vinegar were tested in their original state or in pH neutralized form for efficacy and cytotoxicity in the CCR5-tropic cell-free entry and cell-associated transmission assays, CXCR4-tropic entry and fusion assays, and in a human PBMC-based anti-HIV-1 assay. These products were also tested for their effect on viability of cervico-vaginal cell lines, human cervical explant tissues, and beneficial Lactobacillus species. Results Natural lime and lemon juice and household vinegar demonstrated anti-HIV-1 activity and cytotoxicity in transformed cell lines. Neutralization of the products reduced both anti-HIV-1 activity and cytotoxicity, resulting in a low therapeutic window for both acidic and neutralized formulations. For the natural juices and vinegar, the IC50 was ≤ 3.5 (0.8-3.5)% and the TC50 ≤ 6.3 (1.0-6.3)%. All three liquid products inhibited viability of beneficial Lactobacillus species associated with vaginal health. Comparison of three different toxicity endpoints in the cervical HeLa cell line revealed that all three products affected membrane integrity, cytosolic enzyme release, and dehydrogenase enzyme activity in living cells. The juices and vinegar also exerted strong cytotoxicity in cervico-vaginal cell lines, mainly due to their acidic pH. In human cervical explant tissues, treatment with 5% lemon or lime juice or 6% vinegar induced toxicity similar to application of 100 μg/ml nonoxynol-9, and exposure to 10% lime juice caused tissue damage comparable to treatment with 5% Triton-X-100. Conclusions Lemon and lime juice and household vinegar do not fulfill the safety criteria mandated for a topical microbicide. As a result of their unphysiological formulation for the vaginal tract, they exhibit cytotoxicity to human cell lines, human vaginal tissues, and beneficial vaginal Lactobacillus species.

Depo-Provera (medroxyprogesterone acetate), a long-acting derivative of progesterone, is utilized during many nonhuman primate microbicide studies to facilitate simian immunodeficiency virus (SIV) infection by thinning the vaginal epithelium. To date, the systemic effects of this steroid hormone in regard to SIV/HIV pathogenesis are not well understood, but an increase in infection rates and lymphoproliferation following progesterone application has been reported. Therefore, a proactive study using 20 Chinese rhesus macaques was designed to investigate the effect of a single Depo-Provera injection on SIV disease progression. Group 1 (n = 10) was treated with 30 mg Depo-Provera intramuscularly 30 days prior to intravenous challenge with 50 TCID(50) SIVmac251, while Group 2 (n = 10) remained untreated, but received the same amount of SIV. Blood samples were taken at predetermined intervals to measure RNA viral loads, CD4(+), CD8(+), and CD20(+) lymphocyte counts and percentages and absolute numbers of naive and memory T lymphocytes. Upon statistical endpoint data analysis, none of the parameters measured were shown to be significantly different between the groups. One animal in the Depo-Provera-treated group and two macaques in the control group were euthanized prior to study end due to the development of clinical signs (in weeks 43 and 51, respectively). All other animals were euthanized between weeks 68 and 71 post-SIV infection. Histopathological evaluations revealed that 5 of 10 animals in each group had developed simian AIDS (SAIDS). In summary, this prospective study demonstrated that a single injection of 30 mg Depo-Provera did not have a significant influence on SIV disease progression.

Background We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates. Methods Vaginal and rectal microflora in Chinese rhesus macaques (Macaca mulatta) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization. Results Chinese rhesus macaques harbored abundant vaginal Lactobacillus, with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals. Conclusions The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus-based topical microbicides.

Although many interactions between HIV-1 and human proteins have been reported in the scientific literature, no publicly accessible source for efficiently reviewing this information was available. Therefore, a project was initiated in an attempt to catalogue all published interactions between HIV-1 and human proteins. HIV-related articles in PubMed were used to develop a database containing names, Entrez GeneIDs, and RefSeq protein accession numbers of interacting proteins. Furthermore, brief descriptions of the interactions, PubMed identification numbers of articles describing the interactions, and keywords for searching the interactions were incorporated. Over 100,000 articles were reviewed, resulting in the identification of 1448 human proteins that interact with HIV-1 comprising 2589 unique HIV-1-to-human protein interactions. Preliminary analysis of the extracted data indicates 32% were direct physical interactions (e.g., binding) and 68% were indirect interactions (e.g., up-regulation through activation of signaling pathways). Interestingly, 37% of human proteins in the database were found to interact with more than one HIV-1 protein. For example, the signaling protein mitogen-activated protein kinase 1 has a surprising range of interactions with 10 different HIV-1 proteins. Moreover, large numbers of interactions were published for the HIV-1 regulatory protein Tat and envelope proteins: 30% and 33% of total interactions identified, respectively. The database is accessible at http://www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions/ and is cross-linked to other National Center for Biotechnology Information databases and programs via Entrez Gene. This database represents a unique and continuously updated scientific resource for understanding HIV-1 replication and pathogenesis to assist in accelerating the development of effective therapeutic and vaccine interventions.

Background In many preclinical AIDS research studies, antiretroviral therapy (ART) is administered to experimentally simian immunodeficiency (SIV)-infected rhesus macaques for reduction of viral load to undetectable levels. Prolonged treatment of macaques with a high dose of PMPA (9-[2-(r)-(phosphonomethoxy) propyl] adenine or tenofovir; 30 mg/kg of body weight subcutaneously once daily) can result in proximal renal tubular dysfunction, a Fanconi-like syndrome characterized by glucosuria, aminoaciduria, hypophosphatemia, and bone pathology. In contrast, chronic administration of a low dose of PMPA (10 mg/kg subcutaneously once daily) starting at birth does not seem to be associated with any adverse health effects within 3 years of treatment. In contrast to PMPA, limited information on systemic toxicity in rhesus monkeys is available for FTC (5-fluoro-1-(2R,5S)-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine; emtricitabine) and stavudine (d4T). Results In this study, the clinical and biochemical correlates of tubular nephrosis in SIV-infected rhesus macaques associated with systemic administration of high-dose ART consisting of the three nucleoside analog inhibitors PMPA, FTC, and d4T were investigated. It was found that acute renal failure was uncommon (7.1% of treated animals) and that morphologic evidence of nephropathy, which persisted for more than 300 days following discontinuation of the drug cocktail, was more frequent (52.4% of treated animals). While parameters from single time points lacked predictive value, biochemical alterations in Blood Urea Nitrogen (BUN) and phosphorus were frequently identified longitudinally in the blood of ART-treated animals that developed evidence of nephropathy, and these longitudinal changes correlated with disease severity. Conclusions Recommendations are proposed to limit the impact of drug-induced renal disease in future SIV macaque studies.