In vivo antiviral efficacy of LCTG-002, a pooled, purified human milk secretory IgA product, against SARS-CoV-2 in a murine model of COVID-19.

Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosae, with secretory form (sIgA) being dominant and uniquely stable. sIgA is challenging to produce recombinantly but is naturally found in human milk, which could be considered a global resource for this biologic, justifying its development as a mucosal therapeutic. Presently, SARS-CoV-2 was utilized as a model mucosal pathogen, and methods were developed to efficiently extract human milk sIgA from donors who were naïve to SARS-CoV-2 or had recovered from infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA in their milk (pooled to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1% or greater were all associated with sIgA. Western blot demonstrated that all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher Spike binding (mean endpoint of 0.87 versus 5.87). LCTG-002 was capable of blocking the Spike receptor-binding domain - angiotensin-converting enzyme 2 (ACE2) interaction with significantly greater potency compared to control (mean LCTG-002 IC50 154ug/mL versus 50% inhibition not achieved for control), and exhibited significant neutralization activity against Spike-pseudotyped virus infection (mean LCTG-002 IC50 49.8ug/mL versus 114.5ug/mL for control). LCTG-002 was tested for its capacity to reduce viral lung burden in K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 significantly reduced SARS-CoV-2 titers compared to control when administered at 0.25 mg/day or 1 mg/day, with a maximum TCID50 reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure and efficacious in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics.

In Vivo Antiviral Efficacy of LCTG-002, a Pooled, Purified Human Milk Secretory IgA product, Against SARS-CoV-2 in a Murine Model of COVID-19.

Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosal compartments including the respiratory tract, with the secretory form of IgA (sIgA) being dominant and uniquely stable in these environments. sIgA is naturally found in human milk, which could be considered a global resource for this biologic, justifying the development of human milk sIgA as a dedicated airway therapeutic for respiratory infections such as SARS-CoV-2. In the present study, methods were therefore developed to efficiently extract human milk sIgA from donors who were either immunologically naïve to SARS-CoV-2 (pooled as a control IgA) or had recovered from a PCR-confirmed SARS-CoV-2 infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA Abs in their milk (pooled together to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1.0% or greater were all associated with sIgA. None of the proteins exhibited statistically significant differences between batches. Western blot demonstrated all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher binding to Spike, and was also capable of blocking the Spike - ACE2 interaction in vitro with 6.3x greater potency compared to control IgA (58% inhibition at ∼240ug/mL). LCTG-002 was then tested in vivo for its capacity to reduce viral burden in the lungs of K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 was demonstrated to significantly reduce SARS-CoV-2 titers in the lungs compared to control IgA when administered at either 250ug/day or 1 mg/day, as measured by TCID50, plaque forming units (PFU), and qRT-PCR, with a maximum reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure, efficacious, and well tolerated in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics against SARS-CoV-2 and other mucosal infections.

The Secretory IgA Response in Human Milk Against the SARS-CoV-2 Spike Is Highly Durable and Neutralizing for At Least 1 Year of Lactation Postinfection.

Background: Although in the early pandemic period COVID-19 pathology among young children and infants was typically less severe compared with that observed among adults, this has not remained entirely consistent as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged. There is an enormous body of evidence demonstrating the benefits of human milk antibodies (Abs) in protecting infants against a wide range of enteric and respiratory infections. It is highly plausible that the same holds true for protection against SARS-CoV-2 as this virus infects cells of the gastrointestinal and respiratory mucosae. Understanding the durability of a human milk Ab response over time after infection is critical. Objective: Previously, we examined the Abs present in milk of those recently infected with SARS-CoV-2 and concluded that the response was secretory immunoglobulin A (sIgA) dominant and that these titers were highly correlated with neutralization potency. The present study aimed to monitor the durability of the SARS-CoV-2 IgA and secretory Ab (sAb) response in milk from COVID-19-recovered lactating individuals over 12 months in the absence of vaccination or reinfection. Results: This analysis revealed a robust and durable spike-specific milk sIgA response, and at 9-12 months after infection, 88% of the samples exhibited titers above the positive cutoff for IgA and 94% were above the cutoff for sAb. Fifty percent of participants exhibited less than twofold reduction of spike-specific IgA through 12 months. A strong, significant positive correlation between IgA and sAb against spike persisted throughout the study period. Nucleocapsid-specific Abs were also assessed, which revealed significant background or cross-reactivity of milk IgA against this immunogen, as well as limited/inconsistent durability compared with Spike titers. Conclusion: These data suggest that lactating individuals are likely to continue producing spike-specific Abs in their milk for 1 year or more, which may provide critical passive immunity to infants against SARS-CoV-2 throughout the lactation period.

Assessment of human milk samples obtained pre and post-influenza vaccination reveals a poor boosting of seasonally-relevant, hemagglutinin-specific antibodies.

Introduction: Influenza (flu) vaccination prevented over 100,000 hospitalizations and 7000 deaths from flu over the 2019-2020 season in the USA. Infants <6 months are the most likely to die from flu, though flu vaccines are only licensed for infants >6 months old. Therefore, it is recommended that flu vaccination occur during pregnancy, as this reduces severe complications; however, vaccination rates are suboptimal, and vaccination is also recommended postpartum. For breast/chest-fed infants, the vaccine is believed to elicit protective and robust seasonally-specific milk antibody (Ab). Few comprehensive studies exist examining Ab responses in milk after vaccination, with none measuring secretory Ab (sAb). Determining whether sAbs are elicited is critical, as this Ab class is highly stable in milk and mucosae. Methods: In the present study, our aim was to determine to what extent specific Ab titers in the milk of lactating people were boosted after seasonal influenza vaccination. Over the 2019-2020 and 2020-2021 seasons, milk was obtained pre- and post-vaccination and assessed for specific IgA, IgG, and sAb against relevant hemagglutinin (HA) antigens by a Luminex immunoassay. Results: IgA and sAb were not found to be significantly boosted, while only IgG titers against B/Phuket/3073/2013, included in vaccines since 2015, exhibited an increase. Across the 7 immunogens examined, as many as 54% of samples exhibited no sAb boost. No significant differences for IgA, sAb, or IgG boosting were measured between seasonally-matched versus mismatched milk groups, indicating boosting was not seasonally-specific. No correlations between IgA and sAb increases were found for 6/8 HA antigens. No boost in IgG- or IgA-mediated neutralization post vaccination was observed. Discussion: This study highlights the critical need to redesign influenza vaccines with the lactating population in mind, wherein the aim should be to elicit a potent seasonally-specific sAb response in milk. As such, this population must be included in clinical studies.

The secretory IgA (sIgA) response in human milk against the SARS-CoV-2 Spike is highly durable and neutralizing for at least 1 year of lactation post-infection.

Although in the early pandemic period, COVID-19 pathology among young children and infants was typically less severe compared to that observed among adults, this has not remained entirely consistent as SARS-CoV-2 variants have emerged. There is an enormous body of evidence demonstrating the benefits of human milk antibodies (Abs) in protecting infants against a wide range of enteric and respiratory infections. It is highly plausible that the same holds true for protection against SARS-CoV-2, as this virus infects cells of the gastrointestinal and respiratory mucosae. Understanding the durability of a human milk Ab response over time after infection is critical. Previously, we examined the Abs present in milk of those recently infected with SARS-CoV-2, and concluded that the response was secretory IgA (sIgA)-dominant and that these titers were highly correlated with neutralization potency. The present study aimed to monitor the durability of the SARS-CoV-2 IgA and secretory Ab (sAb) response in milk from COVID-19-recovered lactating individuals over 12 months, in the absence of vaccination or re-infection. This analysis revealed a robust and durable Spike-specific milk sIgA response, that at 9-12 months after infection, 88% of the samples exhibited titers above the positive cutoff for IgA and 94% were above cutoff for sAb. Fifty percent of participants exhibited less than a 2-fold reduction of Spike-specific IgA through 12 months. A strong significant positive correlation between IgA and sAb against Spike persisted throughout the study period. Nucleocapsid-specific Abs were also assessed, which revealed significant background or cross reactivity of milk IgA against this immunogen, as well as limited/inconsistent durability compared to Spike titers. These data suggests that lactating individuals are likely to continue producing Spike-specific Abs in their milk for 1 year or more, which may provide critical passive immunity to infants against SARS-CoV-2 throughout the lactation period.

Qualitative immunoassay for the detection of anti-SARS-COV-2 spike antibody in human milk samples.

Antibodies in milk obtained from those previously SARS-CoV-2-infected or vaccinated against COVID-19 may provide passive immunity to the breastfed infant. Few assays have been established to measure antibodies in human milk, despite the public health importance of this topic. In the present protocol, we describe an optimized indirect ELISA assay aimed to measure SARS-CoV-2-reactive antibodies in human milk, which can be used as a rapid screen on undiluted samples or to designate samples as relatively low, moderate, or high titer. For complete details on the use and execution of this protocol, please refer to Fox et al. (2020).

Safety of breast/chest-feeding by those infected by SARS-CoV-2.

PURPOSE OF REVIEW: One important question from the outset of the pandemic has been whether a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected person's milk might be a vehicle for SARS-CoV-2 transmission. This review summarizes the most recent data on this topic. RECENT FINDINGS: A SARS-CoV-2 sIgA response in milk after infection is very common. To date, there has been no evidence that SARS-CoV-2 transmits via human milk. Though viral RNA has been identified in a minority of milk samples studied, infectious virus particles have not. SUMMARY: The highly dominant transmission route for SARS-CoV-2 is via inhalation of respiratory droplets containing virus particles. Other routes of transmission are possible, including fecal-oral, trans-placental, and to a much lesser extent, via a contaminated surface. SARS-CoV-2 cannot transmit via human milk. There is no evidence that infants should be separated from SARS-CoV-2-infected mothers who are well enough to establish or continue breastfeeding.

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets.

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~105-108 maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Phagocytosis of a Model Human Immunodeficiency Virus Target by Human Breast Milk Leukocytes Is Predominantly Granulocyte-Driven When Elicited by Specific Antibody.

BACKGROUND: Studies demonstrate a protective effect of antibodies (Abs) in breast milk (BM) against mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV). Contribution of the BM cellular component has been overlooked. The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with Abs mediating functions through the constant immunoglobulin region-the crystallizable fragment (Fc). These data support induction of vaccine Abs triggering antiviral activities by leukocytes through Fc receptors (FcRs). OBJECTIVE: To measure Ab-dependent cellular phagocytosis (ADCP), an essential Fc-mediated response, by BM phagocytes. MATERIALS AND METHODS: Cells were isolated from five human BM samples obtained at 7-183 days postpartum and analyzed for ADCP. Fluorescent beads coated with HIV envelope (Env) epitopes were used as targets. Sixty-seven to 100 mL of milk was utilized. RESULTS: Total cell concentrations per milliliter were 16,083-222,857, with 1.6-12.3% being CD45+ leukocytes. ADCP activity was measurable using the HIV-specific Ab 830A. Use of the actin inhibitor cytochalasin D and FcR blocker indicated that ADCP was actin dependent and required FcR engagement. ADCP scores were variable, but largely consistent, across the samples studied, exhibiting <4-fold difference from lowest to highest activity for CD45+ cells. Of the CD45+ ADCP, significantly more activity was granulocyte derived (72-95%), while the remaining activity was monocyte driven. CONCLUSIONS: The data indicate that BM phagocytes can manifest antiviral activities in the presence of specific Abs and therefore may contribute to reduction of MTCT of HIV.

Primary Human Neutrophils Exhibit a Unique HIV-Directed Antibody-Dependent Phagocytosis Profile.

The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with the antibodies (Abs) mediating function via the "constant" immunoglobulin region, the crystallizable fragment (Fc). These data have supported a focus on the induction of Abs by vaccines that trigger antiviral activities by relevant leukocytes via Fc receptors (FcRs). Neutrophils are phagocytes that comprise > 50% of leukocytes and display unique FcRs. We sought to compare the Ab-dependent cellular phagocytosis (ADCP) activity of human neutrophils to the commonly assayed THP-1 cell line. HIV-specific Abs were employed to elicit ADCP of beads coated with HIV envelope protein. Overall, trends were noted among neutrophil donors and the ADCP profile was different from that of THP-1 cells. mAb ELISA titers correlated with ADCP by THP-1 cells but not neutrophils. Monoclonal (m)Abs were also tested with primary monocytes. Donor-to-donor variation was high, and hindered the analysis of this dataset, but it was, in itself, an important finding. This study illustrates the concept that the assessment of FcR-mediated Ab activity with a frequently used cell line such as THP-1 is not necessarily indicative of relevant Ab functionality in vivo, and this calls for in-depth study of the properties of the HIV antibodies best-suited to eliciting antiviral activities by primary cells.

Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer.

We recently showed that mutations in the HIV-1 envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (MAbs). Here, we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wild-type JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 MAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intraprotomer interactions exposed epitopes in the V2i region (which overlies the α4ß7 integrin binding site) and the V3 crown, suggesting general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutations affecting interprotomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed MAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together, these data indicate that several transient Env epitopes can be rendered more accessible to antibodies (Abs) via specific mutations, and this may facilitate the design of V1V2-targeting immunogens.IMPORTANCE Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine trial was correlated with the elicitation of V2- and V3-directed antibodies. Previously, we identified various mechanisms responsible for destabilizing the V3 loop; here, we determined, via mutation of numerous Env residues, which of these elements maintain the V1V2 loop in an inaccessible state and which expose V1V2 and/or V3 epitopes. Notably, our data indicate that V3 can move independently of V2, but none of the mutations studied expose V2 epitopes without also exposing V3. Additionally, V1V2 can be rendered more accessible to Abs via specific mutations, facilitating the development of engineered V2 immunogens.

A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice.

Though vaccination with live-attenuated SIV provides the greatest protection from progressive disease caused by SIV challenge in rhesus macaques, attenuated HIV presents safety concerns as a vaccine; therefore, live viral vectors carrying HIV immunogens must be considered. We have designed a replication-competent vesicular stomatitis virus (VSV) displaying immunogenic HIV-1 Env trimers and attenuating quantities of the native surface glycoprotein (G). The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5'terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG. Western blot analysis indicated that ∼75% of incorporated EnvG was a mature proteolytically processed form. Flow cytometry showed that surface EnvG bound various conformationally- and trimer-specific antibodies (Abs), and in-vitro growth assays on CD4+CCR5+ cells demonstrated EnvG functionality. Neither intranasal (IN) or intramuscular (IM) administration in mice induced any observable pathology and all regimens tested generated potent Env-specific ELISA titers of 10(4)-10(5), with an IM VSV prime/IN VSV boost regimen eliciting the highest binding and neutralizing Ab titers. Significant quantities of Env-specific CD4+ T cells were also detected, which were augmented as much as 70-fold by priming with IM electroporated plasmids encoding EnvG and IL-12. These data suggest that our novel vector can achieve balanced safety and immunogenicity and should be considered as an HIV vaccine platform.

A Multiplex Microsphere-Based Immunoassay Increases the Sensitivity of SIV-Specific Antibody Detection in Serum Samples and Mucosal Specimens Collected from Rhesus Macaques Infected with SIVmac239.

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex(®) Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%-100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.

Rapid, quantitative mapping of anti-HIV type 1 envelope serum antibody specificities.

A new generation of extremely broad and potent neutralizing antibodies (bNAbs) has been isolated from HIV-infected subjects. This has refocused interest in the sites of vulnerability targeted by these bNAbs and in the potential for designing Envelope (Env) immunogens that display these sites. Standard methods for evaluating HIV-1 vaccine candidates do not enable epitope mapping on the HIV Env spike, the target for NAbs. To meet the need for rapid analysis of Ab specificity, we designed a multiplexed, quantitative mapping assay that can test for serum Ab competition for the binding of an HIV-1 Env gp120 to a panel of bNAbs directed to different sites of vulnerability on the Env that do not compete for one another in the assay. Using serum samples from rabbits immunized with various DNA prime/gp120 protein boost vaccines we were able to detect serum Ab competition for multiple classes of bNAbs in the postimmune samples that were significantly higher than background competition detected in samples obtained prior to vaccination. Importantly, application of this novel assay to our ongoing HIV-1 Env viral vector studies in mice has allowed us to distinguish qualitative differences in the Ab elicited by various regimens that ELISA cannot. Furthermore, pooled immunoglobulin from HIV-infected donors (HIVIg) competes for binding to the bNAb panel whereas a control pool from HIV-negative donors does not, highlighting the utility of this assay for human studies. This novel assay will add value in rational immunogen design and in the detailed, qualitative evaluation of binding and, potentially, neutralizing Abs elicited by natural infections and HIV-1 vaccine candidates.

Infection by discordant strains of HIV-1 markedly enhances the neutralizing antibody response against heterologous virus.

High-risk cohorts in East Africa and the United States show rates of dual HIV-1 infection--the concomitant or sequential infection by two HIV-1 strains--of 50% to 100% of those of primary infection, and our normal-risk HIV-positive cohort in Cameroon exhibits a rate of dual infection of 11% per year, signifying that these infections are not exceptional. Little is known regarding the effect of dual infections on host immunity, despite the fact that they provide unique opportunities to investigate how the immune response is affected when challenged with diverse HIV-1 antigens. Using heterologous primary isolates, we have shown here that dual HIV-1 infection by genetically distant strains correlates with significantly increased potency and breadth of the anti-HIV-1 neutralizing antibody response. When the neutralization capacities of sequential plasma obtained before and after the dual infection of 4 subjects were compared to those of matched plasma obtained from 23 singly infected control subjects, a significant increase in the neutralization capacity of the sequential sample was found for 16/28 dually infected plasma/virus pairs, while only 4/159 such combinations for the control subjects exhibited a significant increase (P < 0.0001). Similarly, there was a significant increase in the plasma dilution capable of neutralizing 50% of virus (IC(50)) for 18/24 dually infected plasma/virus pairs, while 0/36 controls exhibited such an increase (P < 0.0001). These results demonstrate that dual HIV-1 infection broadens and strengthens the anti-HIV-1 immune response, suggesting that vaccination schemes that include polyvalent, genetically divergent immunogens may generate highly protective immunity against any HIV-1 challenge strain.

Longitudinal quasispecies analysis of viral variants in HIV type 1 dually infected individuals highlights the importance of sequence identity in viral recombination.

Little is known regarding the likelihood of recombination between any given pair of nonidentical HIV-1 viruses in vivo. The present study analyzes the HIV-1 quasispecies in the C1C2 region of env, the vif-vpr-vpu accessory gene region, and the reverse transcriptase region of pol. These sequences were amplified from samples obtained sequentially over a 12- to 33-month period from five dually HIV-1-infected subjects. Analysis of an average of 248 clones amplified from each subject revealed no recombinants within the three loci studied of the subtype-discordant infecting strains, whose genetic diversity was >11% in env. In contrast, two subjects who were initially coinfected by two subtype-concordant variants with genetic diversity of 7.4% in env were found to harbor 10 unique recombinants of these strains, as exhibited by analysis of the env gene. The frequent recombination observed among the subtype-concordant strains studied herein correlates with prior sequence analyses that have commonly found higher rates of recombination at loci bearing the most conserved sequences, demonstrating an important role for sequence identity in HIV-1 recombination. Viral load analysis revealed that the samples studied contained an average of 8125 virus copies/ml (range, 882-31,626 copies/ml), signifying that the amount of viral RNA in the samples was not limiting for studying virus diversity. These data reveal that recombination between genetically distant strains may not be an immediate or common outcome to dual infection in vivo and suggest critical roles for viral and host factors such as viral fitness, virus diversity, and host immune responses that may contribute to limiting the frequency of intersubtype recombination during in vivo dual infection.

HIV-1 reverse transcriptase drug-resistance mutations in chronically infected individuals receiving or naïve to HAART in Cameroon.

The most common first-line, highly active anti-retroviral therapy (HAART) received by individuals infected with HIV-1 in Cameroon is the combination therapy Triomune, comprised of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-NRTI (NNRTI). To examine the efficacy of these drugs in Cameroon, where diverse non-B HIV-1 subtypes and recombinant viruses predominate, the reverse transcriptase (RT) viral sequences in patient plasma were analyzed for the presence of mutations that confer drug resistance. Forty-nine HIV-1-positive individuals were randomly selected from those receiving care in HIV/AIDS outpatient clinics in the South-West and North-West Regions of Cameroon. Among the 28 patients receiving HAART, 39% (11/28) had resistance to NRTIs, and 46% (13/28) to NNRTIs after a median of 12 months from the start of therapy. Among those with drug-resistance mutations, there was a median of 14 months from the start of HAART, versus 9 months for those without; no difference was observed in the average viral load (10,997 copies/ml vs. 8,056 copies/ml). In contrast, drug-naïve individuals had a significantly higher average viral load (27,929 copies/ml) than those receiving HAART (9,527 copies/ml). Strikingly, among the 21 drug-naïve individuals, 24% harbored viruses with drug-resistance mutations, suggesting that HIV-1 drug-resistant variants are being transmitted in Cameroon. Given the high frequency of resistance mutations among those on first-line HAART, coupled with the high prevalence of HIV-1 variants with drug-resistance mutations among drug-naïve individuals, this study emphasizes the need for extensive monitoring of resistance mutations and the introduction of a second-line HAART strategy in Cameroon.

High frequency of HIV-1 dual infections among HIV-positive individuals in Cameroon, West Central Africa.

OBJECTIVES: To determine the frequency of dual inter- and intra-subtype HIV-1 infection among a cohort of 64 longitudinally-studied, HIV-1-positive individuals in Yaoundé, Cameroon. METHODS: Blood was collected every 3-6 months for up to 36 months and RNA was extracted from plasma. Gag fragment (HxB2 location 1577-2040) was amplified by nested RT-PCR, and mixed-time-point Heteroduplex Assays (HDAs) were performed. As heteroduplexes in this assay indicate >or=5% genetic discordance in the gag fragment, their presence reveals dual infection. Results were confirmed by phylogenetic analysis. RESULTS: Heteroduplexes were generated by specimens of 10 subjects (15.6%). Kaplan-Meier nonparametric estimate of maintenance of single infection was calculated; the rate/year of a 2 infection was found to be approximately 11%. Dual infection was identified in the final specimens of five subjects, after as much as 18 months follow-up, while for the remaining five subjects, dual infection was identified in interim specimens within an average of 10 months follow-up. Analysis of samples obtained after dual infection from each of these latter five subjects revealed two patterns: reversion to initial strain, or replacement of initial strain. Four subjects were dually-infected with HIV-1 strains of the same subtype, while 6 were infected with different subtypes. CONCLUSIONS: The high prevalence of recombinant HIV-1 strains in Cameroon may in part be explained by the high frequency of dual infection. In this genetically-diverse HIV-1 milieu, dual infections and the recombinant viruses they generate are strongly driving viral evolution, complicating vaccine strategies.

A heteroduplex assay for the rapid detection of dual Human Immunodeficiency Virus Type 1 infections.

The predominance of circulating and unique recombinant forms (URFs) of Human Immunodeficiency Virus Type 1 (HIV-1) in Cameroon suggests that dual infection occurs frequently in this region. Despite the potential impact of these infections on the evolution of HIV diversity, relatively few have been detected. The failure to detect dual infections may be attributable to the laborious and costly sequence analysis involved in their identification. As such, there is a need for a cost-effective, more rapid method to efficiently distinguish this subset of HIV-positive individuals, particularly in regions where HIV diversity is broad. In the present study, the heteroduplex assay (HDA) was developed to detect dual HIV-1 infection. This assay was validated on sequential specimens obtained from 20 HIV+ study subjects, whose single or dual infection status was determined by standard sequence analysis. By mixing gag fragments amplified from the sequential specimens from each study subject in HDA reactions, it was shown that single and dual infection status correlated with the absence and presence, respectively, of heteroduplex bands upon gel electrophoresis. Therefore, this novel assay is capable of identifying dual infections with a sensitivity and specificity equivalent to that of sequence analysis. Given the impact of dual infection on viral recombination and diversity, this simple technique will be beneficial to understanding HIV-1 evolution within an individual, as well as at a population level, in West-Central Africa and globally.

Utility of the heteroduplex assay (HDA) as a simple and cost-effective tool for the identification of HIV type 1 dual infections in resource-limited settings.

The predominance of unique recombinant forms (URFs) of HIV-1 in Cameroon suggests that dual infection, the concomitant or sequential infection with genetically distinct HIV-1 strains, occurs frequently in this region; yet, identifying dual infection among large HIV cohorts in local, resource-limited settings is uncommon, since this generally relies on labor-intensive and costly sequencing methods. Consequently, there is a need to develop an effective, cost-efficient method appropriate to the developing world to identify these infections. In the present study, the heteroduplex assay (HDA) was used to verify dual or single infection status, as shown by traditional sequence analysis, for 15 longitudinally sampled study subjects from Cameroon. Heteroduplex formation, indicative of a dual infection, was identified for all five study subjects shown by sequence analysis to be dually infected. Conversely, heteroduplex formation was not detectable for all 10 HDA reactions of the singly infected study subjects. These results suggest that the HDA is a simple yet powerful and inexpensive tool for the detection of both intersubtype and intrasubtype dual infections, and that the HDA harbors significant potential for reliable, high-throughput screening for dual infection. As these infections and the recombinants they generate facilitate leaps in HIV-1 evolution, and may present major challenges for treatment and vaccine design, this assay will be critical for monitoring the continuing pandemic in regions of the world where HIV-1 viral diversity is broad.