The integration of tuberculosis and HIV testing on GeneXpert can substantially improve access and same-day diagnosis and benefit tuberculosis programmes: A diagnostic network optimization analysis in Zambia.

Diagnostic network optimization (DNO), a geospatial optimization technique, can improve access to diagnostics and reduce costs through informing policy-makers' decisions on diagnostic network changes. In Zambia, viral load (VL) testing and early infant diagnosis (EID) for HIV has been performed at centralized laboratories, whilst the TB-programme utilizes a decentralized network of GeneXpert platforms. Recently, the World Health Organization (WHO) has recommended point-of-care (POC) EID/VL to increase timely diagnosis. This analysis modelled the impact of integrating EID/VL testing for children and pregnant/breastfeeding-women (priority-HIV) with TB on GeneXpert in Zambia. Using OptiDx, we established the baseline diagnostic network using inputs for testing demand (October 2019-September 2020), referrals, testing sites, testing platforms, and costs for HIV/TB testing (transport, test, device) respectively in Zambia. Next, we integrated priority-HIV testing on GeneXpert platforms, historically only utilized by the TB-programme. 228,265 TB tests were conducted on GeneXpert devices and 167,458 (99%) of priority-HIV tests on centralized devices at baseline, of which 10% were tested onsite at the site of sample collection. With integration, the average distance travelled by priority-HIV tests decreased 10-fold (98km to 10km) and the proportion tested onsite increased (10% to 48%). 52% of EID tests are likely to be processed within the same-day from a baseline of zero. There were also benefits to the TB-programme: the average distance travelled/specimen decreased (11km to 7km), alongside potential savings in GeneXpert device-operating costs (30%) through cost-sharing with the HIV-programme. The total cost of the combined testing programmes reduced marginally by 1% through integration/optimization. DNO can be used to strategically leverage existing capacity to achieve the WHO's recommendation regarding POC VL/EID testing. Through DNO of the Zambian network, we have shown that TB/HIV testing integration can improve the performance of the diagnostic network and increase the proportion of specimens tested closer to the patient whilst not increasing costs.

Evolution of HIV-1 drug resistance after virological failure of first-line antiretroviral therapy in Lusaka, Zambia.

BACKGROUND: HIV viral load (VL) and resistance testing are limited in sub-Saharan Africa, so individuals may have prolonged time on failing first-line antiretroviral therapy (ART). Our objective was to describe the evolution of drug resistance mutations among adults failing first-line ART in Zambia. METHODS: We analysed data from a trial of VL monitoring in Lusaka, Zambia. From 2006 to 2011, 12 randomized sites provided either routine VL monitoring (intervention) or discretionary (control) after ART initiation. Samples were collected prospectively following the same schedule in each arm but analysed retrospectively in the control group. For those with virological failure (VF; >400 copies/ml), HIV genotyping was performed retrospectively on baseline (BL) and on all subsequent specimens until censored due to study completion, withdrawal or death. RESULTS: Of 1,973 enrollees, 165 (8.4%) developed VF. 464 genotype results were available including 132 (80%) at BL, 116 (70%) at VF and 125 (76%) had at least one result between VF and censoring. Major nucleoside reverse transcriptase inhibitor (NRTI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations increased from 26% (BL) to 82% (VF) to 89% at last genotype (LG). M184 mutations increased from 2% to 59% to 71%; K65R from 2% to 11% to 13%; 2 or more thymidine analogue mutations from 1% to 3% to 12%. Among those on a failing tenofovir disoproxil fumarate (TDF)-based regimen, TDF resistance increased from 42% to 58%. CONCLUSIONS: We found substantial resistance to NRTIs and NNRTIs at VF with incremental increases after VF while still on a failing first-line ART; this resistance may compromise attainment of the UNAIDS 90-90-90 goals.

Neutralization Takes Precedence Over IgG or IgA Isotype-related Functions in Mucosal HIV-1 Antibody-mediated Protection.

HIV-1 infection occurs primarily through mucosal transmission. Application of biologically relevant mucosal models can advance understanding of the functional properties of antibodies that mediate HIV protection, thereby guiding antibody-based vaccine development. Here, we employed a human ex vivo vaginal HIV-1 infection model and a rhesus macaque in vivo intrarectal SHIV challenge model to probe the protective capacity of monoclonal broadly-neutralizing (bnAb) and non-neutralizing Abs (nnAbs) that were functionally modified by isotype switching. For human vaginal explants, we developed a replication-competent, secreted NanoLuc reporter virus system and showed that CD4 binding site bnAbs b12 IgG1 and CH31 IgG1 and IgA2 isoforms potently blocked HIV-1JR-CSF and HIV-1Bal26 infection. However, IgG1 and IgA nnAbs, either alone or together, did not inhibit infection despite the presence of FcR-expressing effector cells in the tissue. In macaques, the CH31 IgG1 and IgA2 isoforms infused before high-dose SHIV challenge were completely to partially protective, respectively, while nnAbs (CH54 IgG1 and CH38 mIgA2) were non-protective. Importantly, in both mucosal models IgG1 isotype bnAbs were more protective than the IgA2 isotypes, attributable in part to greater neutralization activity of the IgG1 variants. These findings underscore the importance of potent bnAb induction as a primary goal of HIV-1 vaccine development.

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

Neutralizing IgG at the portal of infection mediates protection against vaginal simian/human immunodeficiency virus challenge.

Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.

Intrapartum tenofovir and emtricitabine reduces low-concentration drug resistance selected by single-dose nevirapine for perinatal HIV prevention.

A single dose of tenofovir/emtricitabine (TDF/FTC) during labor significantly reduces peripartum nevirapine-associated viral drug resistance when measured by consensus HIV sequencing. It is unknown whether this effect extends to HIV subpopulations of <25-50%. We conducted a randomized trial of single-dose TDF/FTC added to peripartum nevirapine to reduce drug resistance associated with nonnucleoside reverse transcriptase inhibitors (NNRTIs). To detect mutations for NNRTIs comprising > or = 2% of the viral population, we used an oligonucleotide ligation assay (OLA) at codons 103, 106, 181, and 190 of HIV reverse transcriptase. To assess development of drug resistance mutations to our study intervention, OLA was also performed at codons 65 and 184. Among the 328 women included in the 2-week analysis, those receiving TDF/FTC were less likely to have NNRTI resistance by OLA (RR = 0.40, 95% CI = 0.21-0.77). A similar trend was observed among the 315 women included in the 6-week analysis (RR = 0.45, 95% CI = 0.31-0.66). Only two (1%) specimens had detectable K65R by OLA. Both were at 6 weeks postpartum; one was detected in the intervention arm and one in the control arm (p = 0.96). M184V was not detected. The ability of single-dose TDF/FTC to protect against peripartum NVP-induced NNRTI resistance extends to minority populations. This efficacy is achieved without significant selection of TDF- or FTC-resistant viruses.

Role of sindbis virus capsid protein region II in nucleocapsid core assembly and encapsidation of genomic RNA.

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.

Functional requirements of the yellow fever virus capsid protein.

Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

Synthesis of dioxane-based antiviral agents and evaluation of their biological activities as inhibitors of Sindbis virus replication.

The crystal structure of the Sindbis virus capsid protein contains one or two solvent-derived dioxane molecules in the hydrophobic binding pocket. A bis-dioxane antiviral agent was designed by linking the two dioxane molecules with a three-carbon chain having R,R connecting stereochemistry, and a stereospecific synthesis was performed. This resulted in an effective antiviral agent that inhibited Sindbis virus replication with an EC(50) of 14 microM. The synthesis proceeded through an intermediate (R)-2-hydroxymethyl-[1,4]dioxane, which unexpectedly proved to be a more effecting antiviral agent than the target compound, as evidenced by its EC(50) of 3.4 microM as an inhibitor of Sindbis virus replication. Both compounds were not cytotoxic in uninfected BHK cells at concentrations of 1mM.

Design, synthesis, and evaluation of dioxane-based antiviral agents targeted against the Sindbis virus capsid protein.

Dioxane-based antiviral agents targeted to the hydrophobic binding pocket of Sindbis virus capsid protein were designed by computer graphics molecular modeling and synthesized. Virus production using SIN-IRES-Luc and capsid assembly were monitored to evaluate antiviral activity. A compound with a three-carbon linker chain connecting two dioxane moieties inhibited virus production by 50% at a concentration of 40 microM, while (R)-hydroxymethyldioxane inhibited virus production by 50% at a concentration of 1 microM. Both compounds were not cytotoxic in uninfected BHK cells at concentrations of 1mM.