Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results.

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.

Multimodality vaccination against clade C SHIV: partial protection against mucosal challenges with a heterologous tier 2 virus.

We sought to test whether vaccine-induced immune responses could protect rhesus macaques (RMs) against upfront heterologous challenges with an R5 simian-human immunodeficiency virus, SHIV-2873Nip. This SHIV strain exhibits many properties of transmitted HIV-1, such as tier 2 phenotype (relatively difficult to neutralize), exclusive CCR5 tropism, and gradual disease progression in infected RMs. Since no human AIDS vaccine recipient is likely to encounter an HIV-1 strain that exactly matches the immunogens, we immunized the RMs with recombinant Env proteins heterologous to the challenge virus. For induction of immune responses against Gag, Tat, and Nef, we explored a strategy of immunization with overlapping synthetic peptides (OSP). The immune responses against Gag and Tat were finally boosted with recombinant proteins. The vaccinees and a group of ten control animals were given five low-dose intrarectal (i.r.) challenges with SHIV-2873Nip. All controls and seven out of eight vaccinees became systemically infected; there was no significant difference in viremia levels of vaccinees vs. controls. Prevention of viremia was observed in one vaccinee which showed strong boosting of virus-specific cellular immunity during virus exposures. The protected animal showed no challenge virus-specific neutralizing antibodies in the TZM-bl or A3R5 cell-based assays and had low-level ADCC activity after the virus exposures. Microarray data strongly supported a role for cellular immunity in the protected animal. Our study represents a case of protection against heterologous tier 2 SHIV-C by vaccine-induced, virus-specific cellular immune 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.

Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia.

BACKGROUND: We addressed the question whether live-virus challenges could alter vaccine-induced antibody (Ab) responses in vaccinated rhesus macaques (RMs) that completely resisted repeated exposures to R5-tropic simian-human immunodeficiency viruses encoding heterologous HIV clade C envelopes (SHIV-Cs). RESULTS: We examined the Ab responses in aviremic RMs that had been immunized with a multi-component protein vaccine (multimeric HIV-1 gp160, HIV-1 Tat and SIV Gag-Pol particles) and compared anti-Env plasma Ab titers before and after repeated live-virus exposures. Although no viremia was ever detected in these animals, they showed significant increases in anti-gp140 Ab titers after they had encountered live SHIVs. When we investigated the dynamics of anti-Env Ab titers during the immunization and challenge phases further, we detected the expected, vaccine-induced increases of Ab responses about two weeks after the last protein immunization. Remarkably, these titers kept rising during the repeated virus challenges, although no viremia resulted. In contrast, in vaccinated RMs that were not exposed to virus, anti-gp140 Ab titers declined after the peak seen two weeks after the last immunization. These data suggest boosting of pre-existing, vaccine-induced Ab responses as a consequence of repeated live-virus exposures. Next, we screened polyclonal plasma samples from two of the completely protected vaccinees by peptide phage display and designed a strategy that selects for recombinant phages recognized only by Abs present after - but not before - any SHIV challenge. With this "subtractive biopanning" approach, we isolated V3 mimotopes that were only recognized after the animals had been exposed to live virus. By detailed epitope mapping of such anti-V3 Ab responses, we showed that the challenges not only boosted pre-existing binding and neutralizing Ab titers, but also induced Abs targeting neo-antigens presented by the heterologous challenge virus. CONCLUSIONS: Anti-Env Ab responses induced by recombinant protein vaccination were altered by the multiple, live SHIV challenges in vaccinees that had no detectable viral loads. These data may have implications for the interpretation of "vaccine only" responses in clinical vaccine trials.

Novel biopanning strategy to identify epitopes associated with vaccine protection.

Identifying immune correlates of protection is important to develop vaccines against infectious diseases. We designed a novel, universally applicable strategy to profile the antibody (Ab) repertoire of protected vaccine recipients, using recombinant phages encoding random peptide libraries. The new approach, termed "protection-linked (PL) biopanning," probes the Ab paratopes of protected vaccinees versus those with vaccine failure. As proof of concept, we screened plasma samples from vaccinated rhesus macaques (RMs) that had completely resisted multiple mucosal challenges with R5-tropic simian-human immunodeficiency viruses (SHIVs). The animals had been immunized with a multicomponent vaccine (multimeric HIV-1 gp160, HIV-1 Tat, and SIV Gag-Pol particles). After PL biopanning, we analyzed the phagotopes selected for amino acid homologies; in addition to the expected Env mimotopes, one recurring motif reflected the neutralizing Ab epitope at the N terminus (NT) of HIV-1 Tat. Subsequent binding and functional assays indicated that anti-Tat NT Abs were present only in completely or partially protected RMs; peak viremia of the latter was inversely correlated with anti-Tat NT Ab titers. In contrast, highly viremic, unvaccinated controls did not develop detectable Abs against the same epitope. Based upon the protective effect observed in vivo, we suggest that Tat should be included in multicomponent HIV-1 vaccines. Our data highlight the power of the new PL-biopanning strategy to identify Ab responses with significant association to vaccine protection, regardless of the mechanism(s) or targets of the protective Abs. PL biopanning is also unbiased with regard to pathogens or disease model, making it a universal tool.

High-level, lasting antiviral immunity induced by a bimodal AIDS vaccine and boosted by live-virus exposure: prevention of viremia.

OBJECTIVE: To characterize the correlates of protection from systemic infection in a vaccinated rhesus macaque, RAt-9, which had been challenged sequentially with two related clade C simian/human immunodeficiency viruses (SHIV-Cs) yet remained aviremic for more than 5 years despite indirect evidence of cryptic infection. DESIGN: To measure long-term anti-SHIV-C immunity, host genetics and gene-expression patterns for protective correlates. METHODS: Long-term immune reactivity was evaluated and identification of virus in RAt-9 was attempted by RT-PCR analysis of concentrated plasma and blood transfer to CD8(+) cell-depleted infant macaques. Full MHC genotyping of RAt-9, TRIM5α and KIR3DL allelic expression analysis of PBMC, and microarray gene expression analysis were performed. RESULTS: All attempts to detect/isolate virus, including blood transfer to CD8(+) cell-depleted infant rhesus macaques, were negative, and the animal maintained normal levels of memory CD4(+) T cells in both peripheral blood and gut tissues. However, RAt-9 maintained high levels of anti-SHIV-C humoral and cellular immunity, including reactivity to nonvaccine neoantigens (Nef and Rev), up to 63 months postinitial challenge, suggesting chronic sub-threshold infection. RAt-9 expressed the Mamu A*001 allele negative for B*008 and B*017, had a B13 serotype, and had increased expression of killer-cell immunoglobulin-like receptors (KIRs) previously linked to favorable outcomes of lentiviral infection. Elements of the gene expression profiling coincided with genotyping results. RAt-9 also displayed CD8 cell noncytotoxic antiviral response (CNAR) activity. CONCLUSION: Monkey RAt-9 is the first example of a virus-exposed, persistently aviremic animal that has maintained long-term, high-level cellular and humoral antiviral immunity in the absence of an identifiable cryptic reservoir.

Vaccination against heterologous R5 clade C SHIV: prevention of infection and correlates of protection.

A safe, efficacious vaccine is required to stop the AIDS pandemic. Disappointing results from the STEP trial implied a need to include humoral anti-HIV-1 responses, a notion supported by RV144 trial data even though correlates of protection are unknown. We vaccinated rhesus macaques with recombinant simian immunodeficiency virus (SIV) Gag-Pol particles, HIV-1 Tat and trimeric clade C (HIV-C) gp160, which induced cross-neutralizing antibodies (nAbs) and robust cellular immune responses. After five low-dose mucosal challenges with a simian-human immunodeficiency virus (SHIV) that encoded a heterologous R5 HIV-C envelope (22.1% divergence from the gp160 immunogen), 94% of controls became viremic, whereas one third of vaccinees remained virus-free. Upon high-dose SHIV rechallenge, all controls became infected, whereas some vaccinees remained aviremic. Peak viremia was inversely correlated with both cellular immunity (p<0.001) and cross-nAb titers (p<0.001). These data simultaneously linked cellular as well as humoral immune responses with the degree of protection for the first time.

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4ß7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection.

Development of a tier 1 R5 clade C simian-human immunodeficiency virus as a tool to test neutralizing antibody-based immunoprophylaxis.

BACKGROUND: While some recently transmitted HIV clade C (HIV-C) strains exhibited tier 1 neutralization phenotypes, most were tier 2 strains (J Virol 2010; 84:1439). Because induction of neutralizing antibodies (nAbs) through vaccination against tier 2 viruses has proven difficult, we have generated a tier 1, clade C simian-human immunodeficiency virus (SHIV-C) to permit efficacy testing of candidate AIDS vaccines against tier 1 viruses. METHODS: SHIV-1157ipEL was created by swapping env of a late-stage virus with that of a tier 1, early form. RESULTS: After adaptation to rhesus macaques (RM), passaged SHIV-1157ipEL-p replicated vigorously in vitro and in vivo while maintaining R5 tropism. The virus was reproducibly transmissible intrarectally. Phylogenetically, SHIV-1157ipEL-p Env clustered with HIV-C sequences. All RM chronically infected with SHIV-1157ipEL-p developed high nAb titers against autologous as well as heterologous tier 1 strains. CONCLUSIONS: SHIV-1157ipEL-p was reproducibly transmitted in RM, induced cross-clade nAbs, and represents a tool to evaluate anti-HIV-C nAb responses in primates.

R5 clade C SHIV strains with tier 1 or 2 neutralization sensitivity: tools to dissect env evolution and to develop AIDS vaccines in primate models.

BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.

Bimodal AIDS vaccine approach: induction of cellular as well as humoral immunity can protect from systemic infection.

HIV clade C (HIV-C) strains comprise approximately 56% of all HIV infections worldwide, and AIDS vaccines intended for global use must protect against this subtype. Our vaccine strategy has been to induce balanced antiviral immunity consisting of both neutralizing antibody and cell-mediated immune responses, an approach we tested in primates. As reported earlier, after isolating recently transmitted HIV-C strains from Zambian infants, we used env from one such virus, HIV1084i, to generate a multimeric gp160 immunogen. From another virus, isolated from a different child of the same mother-infant cohort, we cloned env to generate a recombinant simian-human immunodeficiency virus (SHIV), which was adapted to rhesus monkeys to yield SHIV-1157ip. Infant macaques were immunized with recombinant viral proteins, including multimeric HIV-C Env 1084i. To test whether cross-protection could be achieved, we mismatched HIV-C Env immunogens and challenge virus env. All vaccinated and control monkeys were exposed orally to low-dose SHIV-1157ip. Animals with no or only transient infection were rechallenged intrarectally with a high dose of R5 SHIV-1157ipd3N4, a "late", animal-evolved variant of SHIV-1157ip. Compared to controls, the vaccinees had significantly lower peak viral RNA loads, and one vaccinee remained completely virus-free, even in lymphoid tissues. Data from our novel heterologous mucosal challenge model and our protein-only immunogens imply that significant protection against heterologous viruses circulating in the local community may be achievable with a strategy that seeks to simultaneously induce cellular immunity as well as neutralizing antibody responses.

Relative transmissibility of an R5 clade C simian-human immunodeficiency virus across different mucosae in macaques parallels the relative risks of sexual HIV-1 transmission in humans via different routes.

BACKGROUND: Worldwide, approximately 90% of all human immunodeficiency virus (HIV) transmissions occur mucosally; almost all involve R5 strains. Risks of sexual HIV acquisition are highest for rectal, then vaginal, and finally oral exposures. METHODS: Mucosal lacerations may affect the rank order of susceptibility to HIV but cannot be assessed in humans. We measured relative virus transmissibility across intact mucosae in macaques using a single stock of SHIV-1157ipd3N4, a simian-human immunodeficiency virus encoding a primary R5 HIV clade C env (SHIV-C). RESULTS: The penetrability of rhesus macaque mucosae differed significantly, with rectal challenge requiring the least virus, followed by vaginal and then oral routes (P = .031, oral vs vaginal; P < .001 rectal vs vaginal). These findings imply that intrinsic mucosal properties are responsible for the differential mucosal permeability. The latter paralleled the rank order reported for humans, with relative risk estimates within the range of epidemiological human studies. To test whether inflammation facilitates virus transmission--as predicted from human studies--we established a macaque model of localized buccal inflammation. Systemic infection occurred across inflamed but not normal buccal mucosa. CONCLUSION: Our primate data recapitulate virus transmission risks observed in humans, thus establishing R5 SHIV-1157ipd3N4 in macaques as a robust model system to study cofactors involved in human mucosal HIV transmission and its prevention.

A comparative study of HIV-1 clade C env evolution in a Zambian infant with an infected rhesus macaque during disease progression.

OBJECTIVE: To evaluate whether HIV-1 clade C (HIV-C) envelope variations that arise during disease progression in rhesus macaque model reflect changes that occur naturally in human infection. DESIGN: An infant macaque was infected with SHIV-1157i, an R5 tropic clade C SHIV, that expresses a primary HIV-C envelope derived from an infected human infant and monitored over a 5-year period. Genetic variation of the V1-V5 envelope region, which is the main target for humoral immune responses, derived from the infected macaque and infant was examined. METHODS: The V1-V5 envelope region was cloned and sequenced from longitudinal peripheral blood mononuclear cell samples collected from the infected macaque and infant. Phylogenetic analysis [phylogenetic tree, diversity, divergence, ratio of nonsynonymous (dN) and synonymous substitution (dS) and dN distribution] was performed. Plasma RNA viral load, CD4(+) T-cell count, changes in the length of V1-V5 region, putative N-linked glycosylation site number and distribution were also measured. RESULTS: Phylogenetic analysis revealed that changes in the macaque closely reflected those of the infant during disease progression. Similar distribution patterns of dN and hot spots were observed between the macaque and infant. Analysis of putative N-linked glycosylation sites revealed several common variations between the virus populations in the two host species. These variations correlate with decline of CD4 T-cell count in the macaque and might be linked with disease progression. CONCLUSION: SHIV-C infection of macaque is a relevant animal model for studying variation of primary HIV-C envelope during disease progression and could be used to analyze the selection pressures that are associated with those changes.

Neutralization-sensitive R5-tropic simian-human immunodeficiency virus SHIV-2873Nip, which carries env isolated from an infant with a recent HIV clade C infection.

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-kappaB 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 moderate [corrected] sensitivity to neutralization led to classification as a tier 2 [corrected] 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.

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.

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

BACKGROUND: Infection 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. RESULTS: We have developed a panel of clade C R5-tropic SHIVs based upon env of a Zambian pediatric isolate of HIV-1 clade C, the world's 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. CONCLUSION: These 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.

Live attenuated Listeria monocytogenes expressing HIV Gag: immunogenicity in rhesus monkeys.

Induction of strong cellular immunity will be important for AIDS vaccine candidates. Natural infection with wild-type Listeria monocytogenes (Lm), an orally transmitted organism, is known to generate strong cellular immunity, thus raising the possibility that live attenuated Lm could serve as a vaccine vector. We sought to examine the potential of live attenuated Lm to induce cellular immune responses to HIV Gag. Rhesus macaques were immunized with Lmdd-gag that expresses HIV gag and lacks two genes in the D-alanine (D-ala) synthesis pathway. Without this key component of the bacterial cell wall, vaccine vector replication critically depends on exogenous D-ala. Lmdd-gag was given to animals either solely orally or by oral priming followed by intramuscular (i.m.) boosting; D-ala was co-administered with all vaccinations. Lmdd-gag and D-ala were well tolerated. Oral priming/oral boosting induced Gag-specific cellular immune responses, whereas oral priming/i.m. boosting induced systemic as well as mucosal anti-Gag antibodies. These results suggest that the route of vaccination may bias anti-Gag immune responses either towards T-helper type 1 (Th1) or Th2 responses; overall, our data show that live attenuated, recombinant Lmdd-gag is safe and immunogenic in primates.

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.

Dendritic cell-based vaccine strategy against human immunodeficiency virus clade C: skewing the immune response toward a helper T cell type 2 profile.

Given the continued spread of human immunodeficiency virus (HIV)-1 worldwide, developing efficient vaccine strategies against HIV-1 is a key task. We tested the safety and immunogenicity of a multicomponent, cell-based vaccine that consisted of antigen-expressing apoptotic bodies with or without autologous dendritic cells (DCs). The vaccine strategy involved transfection of human 293T cells with codon-optimized DNA vectors expressing env of HIV1084i, a newly transmitted pediatric HIV-1 clade C strain; SHIV89.6P tat; and SIVmac239 gag-protease. Apoptotic bodies were generated by heat shock and ultraviolet irradiation and mixed either with mouse DCs (DC-cell vaccine) or given directly (cell-only vaccine) to BALB/c mice for initial priming; boosts consisted of apoptotic bodies only. The immunogens were well tolerated with or without DCs. Compared with the cell-only vaccine, the DC-cell vaccine induced higher antibody titers against all three antigens, whereas virus-specific cytotoxic T lymphocyte responses were equally strong in both groups. Iso-type analysis of viral antigen-specific antibodies revealed a skewing toward helper T type 2 responses induced by the DC-cell vaccine but not by the cell-only vaccine. In summary, both vaccine strategies were safe and induced cellular as well as humoral antiviral immunity; the DC-based approach had the advantage of significantly stronger antibody responses.