Antigen-specific immunoglobulin variable region sequencing measures humoral immune response to vaccination in the equine neonate

<div><p>The value of prophylactic neonatal vaccination is challenged by the interference of passively transferred maternal antibodies and immune competence at birth. Taken our previous studies on equine B cell ontogeny, we hypothesized that the equine neonate generates a diverse immunoglobulin repertoire in response to vaccination, independently of circulating maternal antibodies. In this study, equine neonates were vaccinated with 3 doses of keyhole limpet hemocyanin (KLH) or equine influenza vaccine, and humoral immune responses were assessed using antigen-specific serum antibodies and B cell Ig variable region sequencing. An increase (p<0.0001) in serum KLH-specific IgG level was measured between days 21 and days 28, 35 and 42 in vaccinated foals from non-vaccinated mares. In vaccinated foals from vaccinated mares, serum KLH-specific IgG levels tended to increase at day 42 (p = 0.07). In contrast, serum influenza-specific IgG levels rapidly decreased (p≤0.05) in vaccinated foals from vaccinated mares within the study period. Nevertheless, IGHM and IGHG sequences were detected in KLH- and influenza- sorted B cells of vaccinated foals, independently of maternal vaccination status. Immunoglobulin nucleotide germline identity, IGHV gene usage and CDR length of antigen-specific IGHG sequences in B cells of vaccinated foals revealed a diverse immunoglobulin repertoire with isotype switching that was comparable between groups and to vaccinated mares. The low expression of CD27 memory marker in antigen-specific B cells, and of cytokines in peripheral blood mononuclear cells upon <i>in vitro</i> immunogen stimulation indicated limited lymphocyte population expansion in response to vaccine during the study period.</p></div

Germline identity of antigen-specific IGHG sequences.

<p>The percent of nucleotide germline identity levels of IGHG sequences from KLH-vaccinated foals from non-vaccinated mares (Group A) and KLH-vaccinated foals from KLH-vaccinated mares (Group B) revealed median values of approximately 95%, comparable to that of KLH-specific IGHG sequences sorted from KLH-vaccinated adult mares. Influenza-specific IGHG germline identity levels from influenza-vaccinated foals (Groups C and D) were also near 95%, while those of influenza-vaccinated mares ranged from 85 to 94%. No IGHG sequences could be amplified from non-vaccinated foals (Group E). “ND”, not detected.</p

Germline identity of influenza-specific IGHM sequences.

<p>The percent of germline nucleotide identity of influenza-specific IGHM sequences for each group is presented for day 42. The value for each cloned PCR product is plotted, and the median values are indicated by horizontal lines. The germline identity values of influenza-specific IGHM transcripts from non-vaccinated foals (Group E) were significantly higher than those from influenza-vaccinated foals (Groups C, p = 0.006, and D, p = 0.04).</p

Serum KLH- and influenza-specific IgG levels in vaccinated and non-vaccinated mares.

<p>Mares were vaccinated or non-vaccinated at 6 and 3 weeks prior to their expected foaling date, and antigen-specific IgG levels were measured in serum on day 3 after foaling. A) Serum KLH-specific IgG in vaccinated mares (filled circles) trended toward statistically significant greater levels (p = 0.05) in comparison to non-vaccinated mares (open circles). An outlier result is shown in the non-vaccinated group. B) There was no statistical difference (p>0.05) in the serum influenza-specific IgG levels between the vaccinated (filled circles) and non-vaccinated (open circles) mare groups. Note the presence of pre-existing influenza antibodies in the group of non-vaccinated mares despite lack of vaccination with influenza vaccine in the previous 2 years. The horizontal lines indicate median values.</p

IGHV gene usage in antigen-specific IGHM sequences.

<p>IGHV gene usage in antigen-specific IGHM sequences.</p

Antigen-specific IGHG CDR3 length distribution.

<p>Antigen-specific IGHG CDR3 length distribution.</p

Serum KLH-specific or influenza-specific IgG levels.

<p>Linear mixed model analysis was used to compare antigen-specific IgG levels at each time point for the groups: vaccinated foals from non-vaccinated mares (continuous lines; KLH Group A in filled circles; influenza Group C in open circles), vaccinated foals from vaccinated mares (dashed lines; KLH Group B in filled squares; influenza Group D in open squares); non-vaccinated foals from non-vaccinated mares (dotted lines; KLH Group E in filled triangles, influenza Group E in open triangles). Each symbol represents the least square means estimate generated from the data. Significant differences in serum KLH-specific IgG levels were found on days 28, 35, and 42 between Group A and Group E (filled circles versus filled triangles, p<0.0001, denoted by ***). A trend of increased (p = 0.07) serum KLH-specific IgG levels was also found between KLH-vaccinated foals from vaccinated mares (Group B, filled squares) versus non-vaccinated foals from non-vaccinated mares (Group E, filled triangles) on day 42. For influenza, there were no statistical differences (p>0.05) between vaccinated foals (Groups C (open circles) or D (open squares)) and non-vaccinated foals from non-vaccinated mares (Group E, open triangles) at all time points.</p

Germline identity of KLH-specific IGHM sequences.

<p>The percent of germline nucleotide identity of KLH-specific IGHM sequences for each group is presented for each time point. The value for each cloned PCR product is plotted, and median values are indicated by horizontal lines. No statistically significant differences (p>0.05) were identified over time within a Group or between Groups.</p

IGHV gene usage in antigen-specific IGHG sequences.

<p>IGHV gene usage in antigen-specific IGHG sequences.</p

Quantitative CD38 mRNA expression in antigen-specific foal cells on day 42.

<p>Relative expression of CD38 memory cell marker is shown by groups: KLH-vaccinated foals from non-vaccinated mares (Group A, n = 5), KLH-vaccinated foals from vaccinated mares (Group B, n = 6), influenza-vaccinated foals from non-vaccinated mares (Group C, n = 3), influenza-vaccinated foals from vaccinated mares (Group D, n = 8), and non-vaccinated foals from non-vaccinated mares (Group E, n = 4). Missing data points indicate lack of detectable CD38 mRNA expression. No statistical difference (p>0.05) in percent CD38 mRNA expression was detected between groups.</p