Comparison of Immune Responses to Different Versions of VLP Associated Stabilized RSV Pre-Fusion F Protein

Efforts to develop a vaccine for respiratory syncytial virus (RSV) have primarily focused on the RSV fusion protein. The pre-fusion conformation of this protein induces the most potent neutralizing antibodies and is the focus of recent efforts in vaccine development. Following the first identification of mutations in the RSV F protein (DS-Cav1 mutant protein) that stabilized the pre-fusion conformation, other mutant stabilized pre-fusion F proteins have been described. To determine if there are differences in alternate versions of stabilized pre-fusion F proteins, we explored the use, as vaccine candidates, of virus-like particles (VLPs) containing five different pre-fusion F proteins, including the DS-Cav1 protein. The expression of these five pre-F proteins, their assembly into VLPs, their pre-fusion conformation stability in VLPs, their reactivity with anti-F monoclonal antibodies, and their induction of immune responses after the immunization of mice, were characterized, comparing VLPs containing the DS-Cav1 pre-F protein with VLPs containing four alternative pre-fusion F proteins. The concentrations of anti-F IgG induced by each VLP that blocked the binding of prototype monoclonal antibodies using two different soluble pre-fusion F proteins as targets were measured. Our results indicate that both the conformation and immunogenicity of alternative VLP associated stabilized pre-fusion RSV F proteins are different from those of DS-Cav1 VLPs

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

A Pre-Vaccination Baseline of SARS-CoV-2 Genetic Surveillance and Diversity in the United States

COVID-19 vaccines were first administered on 15 December 2020, marking an important transition point for the spread of SARS-CoV-2 in the United States (U.S.). Prior to this point in time, the virus spread to an almost completely immunologically naïve population, whereas subsequently, vaccine-induced immune pressure and prior infections might be expected to influence viral evolution. Accordingly, we conducted a study to characterize the spread of SARS-CoV-2 in the U.S. pre-vaccination, investigate the depth and uniformity of genetic surveillance during this period, and measure and otherwise characterize changing viral genetic diversity, including by comparison with more recently emergent variants of concern (VOCs). In 2020, SARS-CoV-2 spread across the U.S. in three phases distinguishable by peaks in the numbers of infections and shifting geographical distributions. Virus was genetically sampled during this period at an overall rate of ~1.2%, though there was a substantial mismatch between case rates and genetic sampling nationwide. Viral genetic diversity tripled over this period but remained low in comparison to other widespread RNA virus pathogens, and although 54 amino acid changes were detected at frequencies exceeding 5%, linkage among them was not observed. Based on our collective observations, our analysis supports a targeted strategy for worldwide genetic surveillance as perhaps the most sensitive and efficient means of detecting new VOCs

Quantitation of the latent HIV-1 reservoir from the sequence diversity in viral outgrowth assays

Abstract Background The ability of HIV-1 to integrate into the genomes of quiescent host immune cells, establishing a long-lived latent viral reservoir (LVR), is the primary obstacle to curing these infections. Quantitative viral outgrowth assays (QVOAs) are the gold standard for estimating the size of the replication-competent HIV-1 LVR, measured by the number of infectious units per million (IUPM) cells. QVOAs are time-consuming because they rely on culturing replicate wells to amplify the production of virus antigen or nucleic acid to reproducibly detectable levels. Sequence analysis can reduce the required number of culture wells because the virus genetic diversity within the LVR provides an internal replication and dilution series. Here we develop a Bayesian method to jointly estimate the IUPM and variant frequencies (a measure of clonality) from the sequence diversity of QVOAs. Results Using simulation experiments, we find our Bayesian approach confers significantly greater accuracy over current methods to estimate the IUPM, particularly for reduced numbers of QVOA replicates and/or increasing actual IUPM. Furthermore, we determine that the improvement in accuracy is greater with increasing genetic diversity in the sample population. We contrast results of these different methods applied to new HIV-1 sequence data derived from QVOAs from two individuals with suppressed viral loads from the Rakai Health Sciences Program in Uganda. Conclusions Utilizing sequence variation has the additional benefit of providing information on the contribution of clonality of the LVR, where high clonality (the predominance of a single genetic variant) suggests a role for cell division in the long-term persistence of the reservoir. In addition, our Bayesian approach can be adapted to other limiting dilution assays where positive outcomes can be partitioned by their genetic heterogeneity, such as immune cell populations and other viruses

Defective proviruses rapidly accumulate during acute HIV-1 infection.

Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, human immunodeficiency virus type 1 (HIV-1) persists in CD4(+) T cells in a latent form that is not targeted by the immune system or by ART. This latent reservoir is a major barrier to curing individuals of HIV-1 infection. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective. However, the dynamics of the accumulation and the persistence of defective proviruses during acute HIV-1 infection are largely unknown. Here we show that defective proviruses accumulate rapidly within the first few weeks of infection to make up over 93% of all proviruses, regardless of how early ART is initiated. By using an unbiased method to amplify near-full-length proviral genomes from HIV-1-infected adults treated at different stages of infection, we demonstrate that early initiation of ART limits the size of the reservoir but does not profoundly affect the proviral landscape. This analysis allows us to revise our understanding of the composition of proviral populations and estimate the true reservoir size in individuals who were treated early versus late in infection. Additionally, we demonstrate that common assays for measuring the reservoir do not correlate with reservoir size, as determined by the number of genetically intact proviruses. These findings reveal hurdles that must be overcome to successfully analyze future HIV-1 cure strategies

KK10 and KK10-cross-reactive microbial peptides differentially expand KK10-specific CD8⁺ T cells.

<p>(<b>A</b>) IFN-y release by ELISpot from HIV-positive HLA-B*27⁺ subject PBMCs stimulated with dilutions of KK10 or KK10CR peptides. (<b>B</b>) Characterization of KK10-specific TCR repertoires after PBMC stimulation with KK10 or KK10CR peptides. PBMCs from HIV-1-positive HLA-B*27⁺ subjects were stimulated with peptides for six days, and KK10-specific CD8⁺ T cells were sorted from fresh (unstimulated), KK10-stimulated, or KK10CR-stimulated PBMCs. Frequencies of unique TCR clones as measured by diversity in the TCR-β CDR3 region were quantified by ImmunoSEQ deep sequencing. Each dot shows the frequency of a single TCR clone in the specified KK10-specific CD8⁺ T cell populations. TCR clones that have the same frequency in both populations compared on a plot fall on the diagonal line. Magenta dots indicate TCR clones that are present at significantly different frequencies (minimum 20 reads in either condition, p<0.005, Fisher’s Exact test with Benjamini-Hochberg correction. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192098#pone.0192098.s001" target="_blank">S1 Table</a> shows all associated p-values).</p