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Several human monoclonal Abs for treating Influenza have been evaluated in clinical trials with limited success despite demonstrating superiority in preclinical animal models including mice. To conduct efficacy studies in mice, human monoclonal Abs are genetically engineered to contain mouse heavy chain constant domain to facilitate the engagement of Fc-receptors on mouse immune effector cells. Although studies have consistently reported discrepancies in Ab effectiveness following genetic engineering, the structural and mechanistic basis for these inconsistencies remain uncharacterized. Here, we use homology modeling to predict variable region (VR) analogous monoclonal Abs possessing human IgG1, mouse IgG1, and mouse IgG2a heavy chain constant domains. We then examine predicted 3D structures for variations in the spatial location and orientation of corresponding paratope amino acid residues. By structurally aligning crystal structures of Fabs in complex with hemagglutinin (HA), we show that corresponding paratope amino acid residues for VR-analogous human IgG1, mouse IgG1, and mouse IgG2a monoclonal Abs interact differentially with HA suggesting that their epitopes might not be identical. To demonstrate that variations in the paratope 3D fine architecture have implications for Ab specificity and effectiveness, we genetically engineered VR-analogous human IgG1, human IgG4, mouse IgG1, and mouse IgG2a monoclonal Abs and explored their specificity and effectiveness in protecting MDCK cells from infection by pandemic H1N1 and H3N2 Influenza viruses. We found that VR-analogous monoclonal Abs placed on mouse heavy chain constant domains were more efficacious at protecting MDCK cells from Influenza virus infection relative to those on human heavy chain constant domains. Interestingly, mouse but not human heavy chain constant domains increased target breadth in some monoclonal Abs. These data suggest that heavy chain constant domain sequences play a role in shaping Ab repertoires that go beyond class or sub-class differences in immune effector recruitment. This represents a facet of Ab biology that can potentially be exploited to improve the scope and utilization of current therapeutic or prophylactic candidates for influenza.

The public health threat from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to intensify with emerging variants of concern (VOC) aiming to render COVID-19 vaccines/infection-induced antibodies redundant. The SARS-CoV-2 spike protein is responsible for receptor binding and infection of host cells making it a legitimate antibody target. Antibodies mostly target epitopes in the receptor binding domain (RBD). Mutations occurring within epitopes influence antibody specificity and function by altering their 3D architecture. However, the mechanisms by which non-epitope mutations in the RBD influence antibody specificity and function remain a mystery. We used Protein Data Bank (PDB) deposited 3D structures for the original, Beta, Delta, BA.1, and BA.2 RBD proteins in complex with either neutralizing antibodies or Angiotensin-Converting Enzyme 2 (ACE2) to elucidate the structural and mechanistic basis for neutralizing antibody evasion driven by non-epitope amino acid substitutions in the RBD. Since the mechanism behind the extensively reported functional discrepancies between the same antibody when used individually and when used in an antibody cocktail is lacking, we explored the structural basis for this inconsistency. Finally, since SARS-CoV-2 antibodies are viral mutagens, we deciphered determinants for antibody-pressured amino acid substitutions. On the one hand, we show that non-epitope mutations in the RBD domain of SARS-CoV-2 VOC influence the formation of hydrogen bonds in the paratope-epitope interface by repositioning RBD amino-acid sidechains (AASCs). This increases the distance between complementary donor/acceptor atoms on paratope and epitope AASCs leading to weaker or the complete prevention of the formation of hydrogen bonds in the paratope-epitope interface. On the other hand, we show that SARS-CoV-2 VOC employ the same strategy to simultaneously search for complementary donor/acceptor atoms on ACE2 AASCs to form new interactions, potentially favoring increased viral transmission. Additionally, we illustrate that converting the spike protein to an RBD, a deletion mutation, also repositions epitope AASCs and that AASC interactions in the paratope-epitope interface vary when an antibody is used individually versus when utilized as a cocktail with other antibodies. Finally, we show that the process of substituting immunogenic RBD amino acids begins with the repositioning of their AASCs induced by immune/antibody pressure. We show that donor/acceptor atoms from any amino acid can determine cross-reactivity instead, provided they possess and present spatially pairing donor/acceptor atoms. By studying structural alignments for PDB deposited antibody-RBD 3D structures and relating them to published binding and neutralization profiles of the same antibodies, we demonstrate that minor structural alterations such as epitope AASC repositioning have a major impact on antibody effectiveness and, hence, should receive adequate attention given that protein structure dictates protein function.

SARS-CoV-2-specific antibody responses are engendered in human milk after BNT162b2 vaccination. However, the emergence of variants of concern (VOCs) raises concerns about the specificity of and potential cross-protection mediated by milk antibody responses, which are crucial for passive immunity transferred from breastfeeding mothers to their infants. In this study, we collected milk samples at three different time points pre- and post-vaccination, and measured milk IgA antibody binding to the receptor binding domain (RBD) of the original Wuhan-Hu-1 strain, and the four VOCs, namely Alpha, Beta, Gamma and Delta. We report a significant level of anti-RBD IgA in milk collected at 4–6 weeks after the second dose of vaccination compared to pre-vaccination. We observed around a 30% reduction in binding to most VOCs, including the major circulating Delta variant, compared to the original Wuhan-Hu-1 strain. As COVID-19 vaccines may take some time to be approved for infants, these individuals remain at risk for severe disease and rely mainly on transferred passive immunity. Our findings support the current recommendations for vaccinating lactating women with the aim of transferring mucosal immunity to breastfeeding infants.

The cycling of the trace gas dimethyl sulfide (DMS) and its precursor dimethylsulfoniopropionate (DMSP) may be affected by future ocean acidification and warming. DMSP and DMS concentrations were monitored over 20-days in four mesocosm experiments in which the temperature and pH of coastal water were manipulated to projected values for the year 2100 and 2150. This had no effect on DMSP in the two-initial nutrient-depleted experiments; however, in the two nutrient-amended experiments, warmer temperature combined with lower pH had a more significant effect on DMSP & DMS concentrations than lower pH alone. Overall, this indicates that future warming may have greater influence on DMS production than ocean acidification. The observed reduction in DMSP at warmer temperatures was associated with changes in phytoplankton community and in particular with small flagellate biomass. A small decrease in DMS concentration was measured in the treatments relative to other studies, from −2% in the nutrient-amended low pH treatment to −16% in the year 2150 pH and temperature conditions. Temporal variation was also observed with DMS concentration increasing earlier in the higher temperature treatment. Nutrient availability and community composition should be considered in models of future DMS.

The role of pharmacy in healthcare continues to evolve as pharmacists gain increased clinical responsibilities in the United States, such as the opportunity to prescribe hormonal contraception. Currently, North Carolina (NC) pharmacists do not have this ability. While previous research focused on the perceptions of community pharmacists surrounding this practice, no previous research surveyed all pharmacists in a state. This cross-sectional, web-based survey was distributed to all actively licensed pharmacists residing in the state of NC in November 2018. The primary objective was to determine the likelihood of NC community pharmacists to prescribe hormonal contraception. Secondary outcomes included: evaluation of all respondent support and perceptions of this practice as advocacy occurs on the state organization level and unified support is critical; opinions regarding over-the-counter (OTC) status of contraception; and potential barriers to prescribing. Overall, 83% of community pharmacists were likely to prescribe hormonal contraception. No differences in likelihood to prescribe were detected between geographic settings. Community pharmacists reported that the most common barriers to impact prescribing were added responsibility and liability (69.8%) and time constraints (67.2%). Fewer than 10% of respondents felt that hormonal contraception should be classified as OTC (7.9%). Noncommunity pharmacists were significantly more likely to agree that prescribing hormonal contraception allows pharmacists to practice at a higher level, that increased access to hormonal contraception is an important public health issue, and that rural areas would benefit from pharmacist-prescribed hormonal contraception. Overall, this study found a willingness to prescribe and support from the majority of both community and noncommunity pharmacists. Limitations of the study included a low response rate and potential nonresponse bias. Future research is needed to address solutions to potential barriers and uptake of this practice, if implemented.