Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).

Two Vaccines for Staphylococcus aureus Induce a B-Cell-Mediated Immune Response.

Staphylococcus aureus causes severe disease in humans for which no licensed vaccine exists. A novel S. aureus vaccine (SA4Ag) is in development, targeting the capsular polysaccharides (CPs) and two virulence-associated surface proteins. Vaccine-elicited antibody responses to CPs are efficacious against serious infection by other encapsulated bacteria. Studies of natural S. aureus infection have also shown a role for TH17 and/or TH1 responses in protection. Single-antigen vaccines, including CPs, have not been effective against S. aureus; a multiantigen vaccine approach is likely required. However, the impact of addition of protein antigens on the immune response to CPs has not been studied. Here, the immune response induced by a bivalent CP conjugate vaccine (to model the established mechanism of action of vaccine-induced protection against Gram-positive pathogens) was compared to the response induced by SA4Ag, which contains both CP conjugates and protein antigens, in cynomolgus macaques. Microengraving, flow cytometry, opsonophagocytic assays, and Luminex technology were used to analyze the B-cell, T-cell, functional antibody, and innate immune responses. Both the bivalent CP vaccine and SA4Ag induced cytokine production from naive cells and antigen-specific memory B-cell and functional antibody responses. Increases in levels of circulating, activated T cells were not apparent following vaccination, nor was a TH17 or TH1 response evident. However, our data are consistent with a vaccine-induced recruitment of T follicular helper (TFH) cells to lymph nodes. Collectively, these data suggest that the response to SA4Ag is primarily mediated by B cells and antibodies that abrogate important S. aureus virulence mechanisms.IMPORTANCEStaphylococcus aureus causes severe disease in humans for which no licensed vaccine exists. A novel vaccine is in development that targets multiple elements of the bacteria since single-component vaccines have not shown efficacy to date. How these multiple components alter the immune response raised by the vaccine is not well studied. We found that the addition of two protein components did not alter substantially the antibody responses raised with respect to function or mobilization of B cells. There was also not a substantial change in the activity of T cells, another part of the adaptive response. This study showed that protection by this vaccine may be mediated primarily by antibody protection.

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.