Vaccine induction of heterologous HIV-1-neutralizing antibody B cell lineages in humans.

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.

In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies.

Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

Structural and genetic convergence of HIV-1 neutralizing antibodies in vaccinated non-human primates.

A primary goal of HIV-1 vaccine development is the consistent elicitation of protective, neutralizing antibodies. While highly similar neutralizing antibodies (nAbs) have been isolated from multiple HIV-infected individuals, it is unclear whether vaccination can consistently elicit highly similar nAbs in genetically diverse primates. Here, we show in three outbred rhesus macaques that immunization with Env elicits a genotypically and phenotypically conserved nAb response. From these vaccinated macaques, we isolated four antibody lineages that had commonalities in immunoglobulin variable, diversity, and joining gene segment usage. Atomic-level structures of the antigen binding fragments of the two most similar antibodies showed nearly identical paratopes. The Env binding modes of each of the four vaccine-induced nAbs were distinct from previously known monoclonal HIV-1 neutralizing antibodies, but were nearly identical to each other. The similarities of these antibodies show that the immune system in outbred primates can respond to HIV-1 Env vaccination with a similar structural and genotypic solution for recognizing a particular neutralizing epitope. These results support rational vaccine design for HIV-1 that aims to reproducibly elicit, in genetically diverse primates, nAbs with specific paratope structures capable of binding conserved epitopes.

Cooperation between somatic mutation and germline-encoded residues enables antibody recognition of HIV-1 envelope glycans.

Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline VHDJH rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination.

Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.

Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay.

A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing.

Phenotypic properties of transmitted founder HIV-1.

Defining the virus-host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.

Recombination-mediated escape from primary CD8+ T cells in acute HIV-1 infection.

BACKGROUND: A major immune evasion mechanism of HIV-1 is the accumulation of non-synonymous mutations in and around T cell epitopes, resulting in loss of T cell recognition and virus escape. RESULTS: Here we analyze primary CD8+ T cell responses and virus escape in a HLA B*81 expressing subject who was infected with two T/F viruses from a single donor. In addition to classic escape through non-synonymous mutation/s, we also observed rapid selection of multiple recombinant viruses that conferred escape from T cells specific for two epitopes in Nef. CONCLUSIONS: Our study shows that recombination between multiple T/F viruses provide greater options for acute escape from CD8+ T cell responses than seen in cases of single T/F virus infection. This process may contribute to the rapid disease progression in patients infected by multiple T/F viruses.

Clonal amplification and maternal-infant transmission of nevirapine-resistant HIV-1 variants in breast milk following single-dose nevirapine prophylaxis.

BACKGROUND: Intrapartum administration of single-dose nevirapine (sdNVP) reduces perinatal HIV-1 transmission in resource-limiting settings by half. Yet this strategy has limited effect on subsequent breast milk transmission, making the case for new treatment approaches to extend maternal/infant antiretroviral prophylaxis through the period of lactation. Maternal and transmitted infant HIV-1 variants frequently develop NVP resistance mutations following sdNVP, complicating subsequent treatment/prophylaxis regimens. However, it is not clear whether NVP-resistant viruses are transmitted via breastfeeding or arise de novo in the infant. FINDINGS: We performed a detailed HIV genetic analysis using single genome sequencing to identify the origin of drug-resistant variants in an sdNVP-treated postnatally-transmitting mother-infant pair. Phylogenetic analysis of HIV sequences from the child revealed low-diversity variants indicating infection by a subtype C single transmitted/founder virus that shared full-length sequence identity with a clonally-amplified maternal breast milk virus variant harboring the K103N NVP resistance mutation. CONCLUSION: In this mother/child pair, clonal amplification of maternal NVP-resistant HIV variants present in systemic and mammary gland compartments following intrapartum sdNVP represents one source of transmitted NVP-resistant variants that is responsible for the acquisition of drug resistant virus by the breastfeeding infant. This finding emphasizes the need for combination antiretroviral prophylaxis to prevent mother-to-child HIV transmission.

Postnatally-transmitted HIV-1 Envelope variants have similar neutralization-sensitivity and function to that of nontransmitted breast milk variants.

BACKGROUND: Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n = 13 viruses), five clinically-matched nontransmitting mothers (n = 16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses). RESULTS: There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants. CONCLUSION: Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.

Simultaneous detection of major drug resistance mutations in the protease and reverse transcriptase genes for HIV-1 subtype C by use of a multiplex allele-specific assay.

High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5' end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.

Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/founder genome.

BACKGROUND: A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method. RESULTS: The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region. CONCLUSIONS: These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.

Evolution of drug-resistant viral populations during interruption of antiretroviral therapy.

Analysis of a large number of HIV-1 genomes at multiple time points after antiretroviral treatment (ART) interruption allows determination of the evolution of drug-resistant viruses and viral fitness in vivo in the absence of drug selection pressure. Using a parallel allele-specific sequencing (PASS) assay, potential primary drug-resistant mutations in five individual patients were studied by analyzing over 18,000 viral genomes. A three-phase evolution of drug-resistant viruses was observed after termination of ART. In the first phase, viruses carrying various combinations of multiple-drug-resistant (MDR) mutations predominated with each mutation persisting in relatively stable proportions while the overall number of resistant viruses gradually increased. In the second phase, viruses with linked MDR mutations rapidly became undetectable and single-drug-resistant (SDR) viruses emerged as minority populations while wild-type viruses quickly predominated. In the third phase, low-frequency SDR viruses remained detectable as long as 59 weeks after treatment interruption. Mathematical modeling showed that the loss in relative fitness increased with the number of mutations in each viral genome and that viruses with MDR mutations had lower fitness than viruses with SDR mutations. No single viral genome had seven or more drug resistance mutations, suggesting that such severely mutated viruses were too unfit to be detected or that the resistance gain offered by the seventh mutation did not outweigh its contribution to the overall fitness loss of the virus. These data provide a more comprehensive understanding of evolution and fitness of drug-resistant viruses in vivo and may lead to improved treatment strategies for ART-experienced patients.

Analysis of low-frequency mutations associated with drug resistance to raltegravir before antiretroviral treatment.

Raltegravir is highly efficacious in the treatment of HIV-1 infection. The prevalence and impact on virologic outcome of low-frequency resistant mutations among HIV-1-infected patients not previously treated with raltegravir have not been fully established. Samples from HIV treatment-experienced patients entering a clinical trial of raltegravir treatment were analyzed using a parallel allele-specific sequencing (PASS) assay that assessed six primary and six secondary integrase mutations. Patients who achieved and sustained virologic suppression (success patients, n = 36) and those who experienced virologic rebound (failure patients, n = 35) were compared. Patients who experienced treatment failure had twice as many raltegravir-associated resistance mutations prior to initiating treatment as those who achieved sustained virologic success, but the difference was not statistically significant. The frequency of nearly all detected resistance mutations was less than 1% of viral population, and the frequencies of mutations between the success and failure groups were similar. Expansion of pre-existing mutations (one primary and five secondary) was observed in 16 treatment failure patients in whom minority resistant mutations were detected at baseline, suggesting that they might play a role in the development of drug resistance. Two or more mutations were found in 13 patients (18.3%), but multiple mutations were not present in any single viral genome by linkage analysis. Our study demonstrates that low-frequency primary RAL-resistant mutations were uncommon, while minority secondary RAL-resistant mutations were more frequently detected in patients naïve to raltegravir. Additional studies in larger populations are warranted to fully understand the clinical implications of these mutations.

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys.

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates.

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro , while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo , increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

Accumulated mutations by 6 months of infection collectively render transmitted/founder HIV-1 significantly less fit.

OBJECTIVE: Viral fitness plays an important role in HIV-1 evolution, transmission and pathogenesis. However, how mutations accumulated during early infection affect viral fitness has not been well studied. METHODS: Paired infectious molecular clones (IMCs) for transmitted/founder (T/F) and 6-month (6-mo) viruses post infection were generated from 10 infected individuals to investigate the impact of accumulated mutations on viral fitness by comparing 6-mo viruses to their cognate T/F viruses. RESULTS: All ten 6-mo viruses were less fit than their cognate T/F viruses. Moreover, the fitness losses of the 6-mo viruses correlated with the decrease in viral loads from the peak of viremia. CONCLUSION: These results show that the mutations accumulated during half a year post infection collectively reduce viral fitness and thereby contribute to lowering viral loads.

Streamlined Subpopulation, Subtype, and Recombination Analysis of HIV-1 Half-Genome Sequences Generated by High-Throughput Sequencing.

High-throughput sequencing (HTS) has been widely used to characterize HIV-1 genome sequences. There are no algorithms currently that can directly determine genotype and quasispecies population using short HTS reads generated from long genome sequences without additional software. To establish a robust subpopulation, subtype, and recombination analysis workflow, we amplified the HIV-1 3'-half genome from plasma samples of 65 HIV-1-infected individuals and sequenced the entire amplicon (∼4,500 bp) by HTS. With direct analysis of raw reads using HIVE-hexahedron, we showed that 48% of samples harbored 2 to 13 subpopulations. We identified various subtypes (17 A1s, 4 Bs, 27 Cs, 6 CRF02_AGs, and 11 unique recombinant forms) and defined recombinant breakpoints of 10 recombinants. These results were validated with viral genome sequences generated by single genome sequencing (SGS) or the analysis of consensus sequence of the HTS reads. The HIVE-hexahedron workflow is more sensitive and accurate than just evaluating the consensus sequence and also more cost-effective than SGS.IMPORTANCE The highly recombinogenic nature of human immunodeficiency virus type 1 (HIV-1) leads to recombination and emergence of quasispecies. It is important to reliably identify subpopulations to understand the complexity of a viral population for drug resistance surveillance and vaccine development. High-throughput sequencing (HTS) provides improved resolution over Sanger sequencing for the analysis of heterogeneous viral subpopulations. However, current methods of analysis of HTS reads are unable to fully address accurate population reconstruction. Hence, there is a dire need for a more sensitive, accurate, user-friendly, and cost-effective method to analyze viral quasispecies. For this purpose, we have improved the HIVE-hexahedron algorithm that we previously developed with in silico short sequences to analyze raw HTS short reads. The significance of this study is that our standalone algorithm enables a streamlined analysis of quasispecies, subtype, and recombination patterns from long HIV-1 genome regions without the need of additional sequence analysis tools. Distinct viral populations and recombination patterns identified by HIVE-hexahedron are further validated by comparison with sequences obtained by single genome sequencing (SGS).