Cross-protection and cross-neutralization capacity of ancestral and VOC-matched SARS-CoV-2 adenoviral vector-based vaccines.

COVID-19 vaccines were originally designed based on the ancestral Spike protein, but immune escape of emergent Variants of Concern (VOC) jeopardized their efficacy, warranting variant-proof vaccines. Here, we used preclinical rodent models to establish the cross-protective and cross-neutralizing capacity of adenoviral-vectored vaccines expressing VOC-matched Spike. CoroVaxG.3-D.FR, matched to Delta Plus Spike, displayed the highest levels of nAb to the matched VOC and mismatched variants. Cross-protection against viral infection in aged K18-hACE2 mice showed dramatic differences among the different vaccines. While Delta-targeted vaccines fully protected mice from a challenge with Gamma, a Gamma-based vaccine offered only partial protection to Delta challenge. Administration of CorovaxG.3-D.FR in a prime/boost regimen showed that a booster was able to increase the neutralizing capacity of the sera against all variants and fully protect aged K18-hACE2 mice against Omicron BA.1, as a BA.1-targeted vaccine did. The neutralizing capacity of the sera diminished in all cases against Omicron BA.2 and BA.5. Altogether, the data demonstrate that a booster with a vaccine based on an antigenically distant variant, such as Delta or BA.1, has the potential to protect from a wider range of SARS-CoV-2 lineages, although careful surveillance of breakthrough infections will help to evaluate combination vaccines targeting antigenically divergent variants yet to emerge.

Overview of the SARS-CoV-2 genotypes circulating in Latin America during 2021.

Latin America is one of the regions in which the COVID-19 pandemic has a stronger impact, with more than 72 million reported infections and 1.6 million deaths until June 2022. Since this region is ecologically diverse and is affected by enormous social inequalities, efforts to identify genomic patterns of the circulating SARS-CoV-2 genotypes are necessary for the suitable management of the pandemic. To contribute to the genomic surveillance of the SARS-CoV-2 in Latin America, we extended the number of SARS-CoV-2 genomes available from the region by sequencing and analyzing the viral genome from COVID-19 patients from seven countries (Argentina, Brazil, Costa Rica, Colombia, Mexico, Bolivia, and Peru). Subsequently, we analyzed the genomes circulating mainly during 2021 including records from GISAID database from Latin America. A total of 1,534 genome sequences were generated from seven countries, demonstrating the laboratory and bioinformatics capabilities for genomic surveillance of pathogens that have been developed locally. For Latin America, patterns regarding several variants associated with multiple re-introductions, a relatively low percentage of sequenced samples, as well as an increment in the mutation frequency since the beginning of the pandemic, are in line with worldwide data. Besides, some variants of concern (VOC) and variants of interest (VOI) such as Gamma, Mu and Lambda, and at least 83 other lineages have predominated locally with a country-specific enrichments. This work has contributed to the understanding of the dynamics of the pandemic in Latin America as part of the local and international efforts to achieve timely genomic surveillance of SARS-CoV-2.

Infants Younger Than 6 Months Infected With SARS-CoV-2 Show the Highest Respiratory Viral Loads.

There is a paucity of reports on the characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in infants, because most studies have grouped infants with older children. We analyzed the viral loads of 45318 SARS-CoV-2-positive nasopharyngeal swab samples obtained in Buenos Aires, Argentina. Infants younger than 6 months presented higher viral loads than any other age group. Children older than 6 months showed significantly lower viral loads, similar to those founds in adults. This observation raises new questions regarding the role of infants in the spreading of SARS-CoV-2 infection.

MicroRNAs differentially present in the plasma of HIV elite controllers reduce HIV infection in vitro.

Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.

HIV, HBV, and HCV molecular epidemiology among trans (transvestites, transsexuals, and transgender) sex workers in Argentina.

Commercial sex work is frequent among male-to-female transvestites, transsexuals and transgenders in Argentina, leading to high susceptibility to HIV, HBV, and HCV among other sexually transmitted infections. In a global context of scarce data on the trans sex workers population, this study was aimed to study the genomic characterization of these viruses. Plasma presence of HIV, HBV, and HCV genomic material was evaluated in samples from 273 trans sex workers. Genomic sequences of HIV-gag, pol, and vif-vpu genes, HBV-S gene, and HCV-5'UT and NS5B genes were obtained. Molecular characterization involved phylogenetic analysis and several in silico tools. Resistance-associated mutations in HIV and HBV pol genes were also analyzed. The HIV genomic characterization in 62 trans sex workers samples showed that 54.8% of the isolates corresponded to BF intersubtype recombinants, and 38.7% to subtype B. The remaining were classified as subtypes C (4.8%) and A (1.6%). HBV and HCV co-infection prevalence among HIV positive trans sex workers yielded rates of 3.2% and 6.5% respectively. Drug resistance-associated mutations were found in 12/62 (19%) HIV pol sequences, but none among HBV. Based on phylogenetic relationships, HIV isolates characterized as subtypes BF and B appeared intermingled with those from other high-risk groups. Despite trans sex workers declared not to have received antiviral treatment, complex drug resistance-associated mutation patterns were found in several HIV isolates. Planned prevention, screening, and treatment are needed to reduce further transmission and morbidity.

Human immunodeficiency virus-1 BF intersubtype recombinant viral protein U second α helix plays an important role in viral release and BST-2 degradation.

We previously reported a naturally occurring BF intersubtype recombinant viral protein U (Vpu) variant with an augmented capacity to enhance viral replication. Structural analysis of this variant revealed that its transmembrane domain and α-helix I in the cytoplasmic domain (CTD) corresponded to subtype B, whereas the α-helix II in the CTD corresponded to subtype F1. In this study, we aimed to evaluate the role of the Vpu cytoplasmic α-helix II domain in viral release enhancement and in the down-modulation of BST-2 and CD4 from the cell surface. In addition, as serine residues in Vpu amino acid positions 61 or 64 have been shown to regulate Vpu intracellular half-life, which in turn could influence the magnitude of viral release, we also studied the impact of these residues on the VpuBF functions, since S61 and S64 are infrequently found among BF recombinant Vpu variants. Our results showed that the exchange of Vpu α-helix II between subtypes (B→F) directly correlated with the enhancement of viral release and, to a lesser extent, with changes in the capacity of the resulting chimera to down-modulate BST-2 and CD4. No differences in viral release and BST-2 down-modulation were observed between VpuBF and VpuBF-E61S. On the other hand, VpuBF-A64S showed a slightly reduced capacity to enhance viral production, but was modestly more efficient than VpuBF in down-modulating BST-2. In summary, our observations clearly indicate that α-helix II is actively involved in Vpu viral-release-promoting activity and that intersubtype recombination between subtypes B and F1 created a protein variant with a higher potential to boost the spread of the recombinant strain that harbours it.

HIV-mediated up-regulation of invariant chain (CD74) correlates with generalized immune activation in HIV+ subjects.

HIV Nef-mediated up-regulation of invariant chain (Ii chain, also CD74) is presumed to play an active role in HIV immunopathogenesis. However, this has not been definitely ascertained. In order to help elucidate this hypothesis, Ii chain, CD4, HLA-DR and HLA-ABC expression was analyzed ex vivo in monocyte-derived macrophages (MDMs) from HIV(+) subjects. Viral load, CD4(+) T cell count and immune activation were also determined in enrolled subjects. Correlations between these parameters and the modulation of cell surface molecules in infected cells were studied. Ii chain expression was found to be up-regulated in infected MDMs derived from all patients but one (median fold up-regulation 2.47±1.82 (range 0.87-7.36)). Moreover, the magnitude of Ii chain up-regulation significantly correlated with higher activation of B and CD4(+) T cells (studied by HLA-DR and CD38 expression). On the other hand, lower HLA-ABC (i.e. stronger down-regulation) in infected MDMs was associated with higher CD4 counts. No correlation was observed between the magnitude of Ii chain up-regulation and the other Nef functions studied here. This is the first study reporting that Ii chain up-regulation occurs on naturally infected antigen presenting cells obtained directly from HIV(+) subjects. Moreover, it is also shown that the magnitude of this up-regulation correlates with immune activation. This allows postulating an alternative hypothesis regarding the contribution of Ii chain up-regulation to HIV-mediated immune damage.

T-cell immune responses against Env from CRF12_BF and subtype B HIV-1 show high clade-specificity that can be overridden by multiclade immunizations.

BACKGROUND: The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors. METHODOLOGY/PRINCIPAL FINDINGS: As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B. CONCLUSIONS/SIGNIFICANCE: This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.

Viral replication is enhanced by an HIV-1 intersubtype recombination-derived Vpu protein.

BACKGROUND: Multiple HIV-1 intersubtype recombinants have been identified in human populations. Previous studies from our lab group have shown that the epidemic in Argentina is characterized by the high prevalence of a circulating recombinant form, CRF12_BF, and many related BF recombinant forms. In these genomic structures a recombination breakpoint frequently involved the vpu coding region. Due to the scarce knowledge of Vpu participation in the virion release process and its impact on pathogenesis and of the functional capacities of intersubtype recombinant Vpu proteins, the aim of this work was to perform a comparative analysis on virion release capacity and relative replication capacity among viral variants harboring either a BF recombinant Vpu or a subtype B Vpu. RESULTS: Our results showed that BF recombinant Vpu was associated to an increased viral particles production when compared to WT B variant in tetherin-expressing cell lines. This observation was tested in the context of a competition assay between the above mentioned variants. The results showed that the replication of the BF Vpu-harboring variant was more efficient in cell cultures than subtype B, reaching a higher frequency in the viral population in a short period of time. CONCLUSION: This study showed that as a result of intersubtype recombination, a structurally re-organized HIV-1 Vpu has an improved in vitro capacity of enhancing viral replication, and provides evidence of the changes occurring in this protein function that could play an important role in the successful spread of intersubtype recombinant variants.

In vitro dynamics of HIV-1 BF intersubtype recombinants genomic regions involved in the regulation of gene expression.

HIV-1 intersubtype recombination is a very common phenomenon that has been shown to frequently affect different viral genomic regions. Vpr and Tat are viral proteins known to interact with viral promoter (LTR) during the replication cycle. This interaction is mainly involved in the regulation of viral gene expression, so, any structural changes in the LTR and/or these regulatory proteins may have an important impact on viral replication and spread. It has been reported that these genetic structures underwent recombination in BF variants widely spread in South America. To gain more insight of the consequences of the BF intersubtype recombination phenomenon on these different but functionally related genomic regions we designed and performed and in vitro study that allowed the detection and recovery of intersubtype recombinants sequences and its subsequent analysis. Our results indicate that recombination affects differentially these regions, showing evidence of a time-space relationship between the changes observed in the viral promoter and the ones observed in the Vpr/Tat coding region. This supports the idea of intersubtype recombination as a mechanism that promotes biological adaptation and compensates fitness variations.

Single Nef proteins from HIV type 1 subtypes C and F fail to upregulate invariant chain cell surface expression but are active for other functions.

HIV-1 Nef protein plays a major role in viral immunopathogenesis, modulating surface expression of several immune receptors, altering signal transduction pathways, and enhancing viral infectivity, among other activities. Nef also exhibits great intersubtype diversity, but most studies have been focused only on Nef proteins from subtype B. Thus, little is known about the functional capacities of nonsubtype B Nef proteins in host cells. Here, we investigated cell surface regulation of MHC-I, MHC-II, the MHC-II-associated chaperone invariant chain (Ii), CD4, CD3, and CD28 in cells transfected or infected with five different Nef alleles including one HIV-1 subtype C and F allele. No significant difference among the Nef proteins regarding CD3, CD28, and MHC-II downregulation was observed. The NefC showed a slightly, yet significant, diminished capacity to downregulate MHC-I in all cells, as well as to downregulate CD4 in Jurkat cells and PBMCs. Strikingly, the two alleles from NefC and NefF were unable to upregulate the Ii chain both in transfected and infected cells. Moreover, the internalization rate of the surface Ii chain was only slightly affected by NefC and NefF, whereas it was drastically reduced by NefB. Nef domains known to be involved in Ii chain upregulation were conserved among the five alleles analyzed here. In summary, we identified two primary HIV-1 NefC and NefF alleles that are selectively impaired for Ii upregulation and that may help to elucidate the mechanism of this Nef function in the future. It will be important to determine whether the observed differences are HIV-1 subtype dependent and influence viral immunopathogenesis.

Drug-resistance surveillance among newly HIV-1 diagnosed individuals in Buenos Aires, Argentina.

OBJECTIVE: Our objective was to estimate primary resistance in an urban setting in a developing country with a long history of antiretroviral delivery and high coverage levels. DESIGN: We carried out a resistance surveillance study according to WHO HIV-Resistance Guidelines. METHODS: Blood samples were collected from 323 drug-naive HIV-1 infected individuals diagnosed at two HIV voluntary counselling and testing centers in Buenos Aires. Viral-load, CD4 cell counts and detuned assays were performed on all samples. The pol gene was sequenced and the resistance profile determined. Phylogenetic analysis was performed by neighbor-joining trees and bootscanning analysis. RESULTS: We found that 12 (4.2%) of the 284 samples sequenced harbored primary resistance mutations, of which K103N, M41L and V108I were most prevalent. Phylogenetic analysis revealed evidence for the transmission of the K103N mutation among the drug-naive population. The proportion of recent infections identified by the detuned assay was 10.1%. CONCLUSIONS: Levels of primary resistance in Buenos Aires are still low, despite a long history of ARV delivery and high coverage levels.

Use of a novel assay based on intact recombinant viruses expressing green (EGFP) or red (DsRed2) fluorescent proteins to examine the contribution of pol and env genes to overall HIV-1 replicative fitness.

Multiple studies have described a reduction in the replicative fitness of HIV-1 isolates harboring mutations that confer resistance to antiretroviral drugs. Contradictory results, however, have been obtained depending on the methodology used in each study (Quinones-Mateu, M.E., Arts, E.J., 2002. Fitness of drug resistant HIV-I: methodology and clinical implications. Drug Resist. Update 5, 224-233), affecting our understanding of the potential relationship of viral replicative fitness with HIV-1 disease. It has been demonstrated previously that both pol and env genes play a major role in HIV-1 replicative fitness of clinical isolates. Therefore, measuring clinically relevant replicative fitness using recombinant viruses where a single mutation and/or viral gene have been introduced does not seem like a reasonable approach in this era of multi-target antiretroviral therapy. A novel method was developed to measure HIV-1 replicative fitness based on recombinant viruses expressing the enhanced green fluorescent (EGFP) or the Discosoma sp. red fluorescent (DsRed2) proteins in a HIV-1NL4-3 backbone. Contrary to previous designs to analyze HIV-1 fitness, these replication competent viruses were created in an intact viral genetic background (without deleting or affecting the expression of any viral gene). This new system was used to evaluate the contribution of drug-resistance mutations in the pol and env genes to overall viral replicative fitness (in the presence and absence of drug pressure) using direct growth competition experiments. Mutations in pol showed a stronger effect on HIV-1 replicative fitness than mutations in the env gene associated with resistance to enfuvirtide, corroborating the plasticity of the later gene to accept mutations and the sensibility of the protease and reverse transcriptase enzymes to drug-associated primary mutations. In conclusion, a new protocol was used to measure HIV-1 replicative fitness in either the presence or absence of antiretroviral drugs, which may be used as a high-throughput assay to help us understand the clinical significance of viral fitness.