Regulation of B cell response to respiratory viruses

Viruses replicating in the respiratory tract (RT) triggers a wide- range of cytokines and chemokines that have antiviral and pro-inflammatory features, instigating an efficient virus- specific B and T cell response that aids in virus- clearance. The majority of antibody secreting cells (ASCs) localizing in the upper RT secrete IgA that can effectively neutralize viruses. In addition, elements of B cell memory are generated that can provide protection from re-infection. Studies examining these aspects, following murine gammaherpesvirus 68 (MHV-68) infection comprise chapter 2 of the dissertation work. Our studies demonstrate that following MHV-68 infection, unlike influenza infection, resulted in a generalized deficiency of virus-specific IgA induction and deficient B cell memory establishment in the respiratory tract. The studies indicate that these aspects of B cell response are regulated by features of virus- replication in the RT. These studies lead to the speculation that these features of B cell response may represent an evolutionary adaptation of viruses that establish long-term latency and are transmitted periodically after reactivation and shedding in secretions. Following cognate interactions with CD4+ T cells, the B cells undergo proliferation, isotype-switching and differentiate towards extrafollicular (low affinity, rapid) or germinal center pathway (high affinity). It is not clear what factors regulate these pathways of B cell differentiation, especially in the context of virus infection in the RT. Studies examining these aspects following influenza infection comprise chapter 3 of the dissertation work. Our studies establish a model for the investigation of host and viral factors that modulate the quality and effectiveness of the B cell response to influenza infection. The findings indicate that the strength of the extrafollicular B cell response depends on the nature of the infecting virus. We present evidence that this pathway of rapid antiviral antibody production relates to the production of non-specifically acting factors in the lung and also dependent of the cytokine profile of virus-specific CD4+T cells. In summary, the current dissertation findings point out to an influence of virus and host associated factors in regulating features of B cell response in the RT

Host Differences in Influenza-Specific CD4 T Cell and B Cell Responses Are Modulated by Viral Strain and Route of Immunization

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses

Impact of COVID-19 on women and children and the need for a gendered approach in vaccine development

The COVID-19 pandemic has imposed unprecedented health and socioeconomic challenges on public health, disrupting it on a global scale. Given that women and children are widely considered the most vulnerable in the times of emergency, whether in war or during a pandemic, the current pandemic has also severely disrupted access to reproductive and child health services. Despite this, data on the effect of the pandemic on pregnant women and newborns remain scarce, and gender-disaggregated indicators of mortality and morbidity are not available. In this context, we suggest the implementation of a gendered approach to ensure the specific needs of women and their newborns are considered during the development of COVID-19 vaccines. Taking into account gender-based biological differences, the inclusion of pregnant and lactating mothers in clinical trials for the development of COVID-19 vaccines is of vital importance

The B cell response following infection with different viruses that replicate in the lung.

<p>(A–F) Virus-specific ASC frequencies. DR1 (A–C) and B10 (D–F) mice were infected intranasally with the influenza viruses PR8 (A and D) and X31 (B and E), and with the non-influenza virus MHV68 (C and F). Virus-specific ASC frequencies in the MedLN were determined by ELISPOT assay at intervals after infection. (G) PR8 replication in the lung. Titers are represented as log₁₀ TCID₅₀/0.2 ml of lung homogenate. (H, I) Flow cytometric analysis of ASCs and germinal center B cells in the MedLN on day 8 after PR8 infection. ASC frequencies (H) represent the proportion of B220^int CD138⁺ cells after gating on live CD4⁻ CD8⁻ CD19⁺ cells. Germinal center B cell frequencies (I) represent the proportion of PNA⁺ Fas⁺ cells among live CD4⁻ CD8⁻ B220⁺ cells. (J) Serum levels of virus-specific IgG in DR1 and B10 mice on day 8 after PR8 infection. Titers determined by ELISA are shown as the reciprocal of the highest serum dilution scored as positive relative to naïve control serum. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone-0034377-g006" target="_blank">Fig. 6</a> data sets depict the mean+SE for 3–10 individual mice per group. * P<0.05, *** P<0.001.</p

The virus-specific B cell and CD4 T cell response to intramuscular immunization.

<p>(A–D) Virus-specific ASC frequencies. DR1 (A and B) and B10 (C and D) mice were immunized intramuscularly with inactivated NC. Virus-specific ASC frequencies in the iliac lymph nodes and spleen were determined by ELISpot assay at the indicated times after immunization. The mean+SE is shown for 3–4 individual mice per group. (E, F) Cytokine production by peptide-specific CD4 T cells. Enriched CD4 T cells from the spleen were analyzed on day 8 after immunization. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and sets of 1–3 17-mer peptides from different viral proteins (HA, NA, NP, and M1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean ± range for 2 independent experiments (the M1 protein was represented in only one experiment with B10 mice). Cells from at least three mice were pooled for each experiment. (G, H) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in E and F. The mean ± range is shown for the two independent experiments.</p

Serum levels of virus-specific Ab in DR1 and B10 mice after NC infection.

<p>NC-specific IgM (A), IgG1 (B), IgG2b (C), IgG2c (D), IgG3 (E), IgA (F), IgG (G), and Ig (H) titers were determined by ELISA on plates coated with disrupted viral particles. Titers are shown as the reciprocal of the highest serum dilution scored as positive relative to naïve control serum. The mean+SE is shown for 4–8 individual mice per group. * P<0.05, ** P<0.01.</p

The CD4 T cell response to PR8 infection.

<p>(A, B) Cytokine production by peptide-specific CD4 T cells. DR1 and B10 mice were infected intranasally with PR8. Enriched CD4 T cells from the MedLN were analyzed on day 10 after infection. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and individual 17-mer peptides. Peptide designations (x-axis) include the viral proteins of origin (NP, M1, and NS1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean ± range for 2 independent experiments. Cells from at least three mice were pooled for each experiment. (C, D) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in A and B. The mean ± range is shown for the two independent experiments.</p

The CD4 T cell response to NC infection.

<p>(A–D) Cytokine production by peptide-specific CD4 T cells. DR1 (A and B) and B10 (C and D) mice were infected intranasally with 40,000 EID₅₀ NC. Enriched CD4 T cells from the MedLN and spleen were analyzed on day 10 after infection. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and individual 17-mer peptides. Peptide designations (x-axis) include the viral proteins of origin (HA, NA, NP, M1, and NS1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean+SEM for 2–6 independent experiments for each peptide. Cells from at least three mice were pooled for each experiment. (E, F) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in A–D and represent 4 (MedLN) or 6 (spleen) independent experiments evaluating 3–12 (MedLN) or 12–20 (spleen) individual peptides. The mean+SEM is shown.</p