A replication-competent adenovirus-vectored influenza vaccine induces durable systemic and mucosal immunity

Abstract

BACKGROUNDTo understand the features of a replicating vaccine that might drive potent and durable immune responses to transgene-encoded antigens, we tested a replication-competent adenovirus type 4 encoding influenza virus H5 HA (Ad4-H5-Vtn) administered as an oral capsule or via tonsillar swab or nasal spray.METHODSViral shedding from the nose, mouth, and rectum was measured by PCR and culturing. H5-specific IgG and IgA antibodies were measured by bead array binding assays. Serum antibodies were measured by a pseudovirus entry inhibition, microneutralization, and HA inhibition assays.RESULTSAd4-H5-Vtn DNA was shed from most upper respiratory tract-immunized (URT-immunized) volunteers for 2 to 4 weeks, but cultured from only 60% of participants, with a median duration of 1 day. Ad4-H5-Vtn vaccination induced increases in H5-specific CD4+ and CD8+ T cells in the peripheral blood as well as increases in IgG and IgA in nasal, cervical, and rectal secretions. URT immunizations induced high levels of serum neutralizing antibodies (NAbs) against H5 that remained stable out to week 26. The duration of viral shedding correlated with the magnitude of the NAb response at week 26. Adverse events (AEs) were mild, and peak NAb titers were associated with overall AE frequency and duration. Serum NAb titers could be boosted to very high levels 2 to 5 years after Ad4-H5-Vtn vaccination with recombinant H5 or inactivated split H5N1 vaccine.CONCLUSIONReplicating Ad4 delivered to the URT caused prolonged exposure to antigen, drove durable systemic and mucosal immunity, and proved to be a promising platform for the induction of immunity against viral surface glycoprotein targets.TRIAL REGISTRATIONClinicalTrials.gov NCT01443936 and NCT01806909.FUNDINGIntramural and Extramural Research Programs of the NIAID, NIH (U19 AI109946) and the Centers of Excellence for Influenza Research and Surveillance (CEIRS), NIAID, NIH (contract HHSN272201400008C).

Keywords: Adaptive immunity; Beta cells; Immunology; Influenza; Vaccines.

Conflict of interest statement

Conflict of interest: MC is an inventor on a patent titled “Replication-competent adenovirus type 4 SAR-CoV-2 vaccines and their use (patent application no. 63/138,221). MC, JS, and JA are inventors on a patent titled “Replication-competent adenovirus type 4–HIV Env vaccines and their use” (patent application no. 63/063,810).

Figures

Figure 1. Ad4-H5-Vtn shedding at mucosal sites.

(A) qPCR was performed on the nasal, tonsillar, and rectal swabs for the detection of viral shedding up to 4 weeks (28 days) after vaccination. Immunization is shown for intranasal (red, n = 25), tonsillar (blue, n = 28), and oral (black, n = 10) routes. (B) Recovery of infectious virus (green) on nasal swabs was measured in comparison with qPCR (gray) in vaccinees immunized by the intranasal route (left). The distribution of days of shedding (middle) and the titers of recovered virus over time (right) are shown (n = 25). The red horizontal bar indicates the median value.

Figure 2. Induction of H5-specific cellular and humoral responses following immunization with Ad4-H5-Vtn.

(A) H5-specific CD4+ and CD8+ T cell responses measured after a 6-hour stimulation with an A/Vietnam/1203/2004 (H5N1) influenza virus peptide array are shown for samples taken at baseline (week 0 [W0]) and 4 weeks (W4) after immunization with the Ad4-H5-Vtn vaccine, which had been administered as an oral capsule (n = 9), by direct tonsillar application (n = 20), or by intranasal spray (n = 25). In addition, responses for individuals who were Ad4 seropositive prior to vaccination are shown as separate exploratory arm (XA) subgroups (Tonsillar XA, n = 4; Intranasal XA, n = 5). Background responses to media controls have been subtracted. Paired and unpaired 2-sample t tests were used for group comparisons. Only significant P values are shown. (B) Ad4 (oral, n = 9; tonsillar, n = 22; intranasal, n = 24) and H5 (oral, n = 7; tonsillar, n = 16; intranasal, n = 20; individuals with less than a 4-fold increase in Ad4 and H5 neutralization [neut.] titer after immunization were excluded) HA-specific neutralizing antibody titers, shown as ID50, were measured by a PVEI assay. The XA subgroups were excluded. Red horizontal bars indicate the median values. Generalized estimating equations with an autoregressive correlation structure were used to calculate P values (week 0 measurements were not included), and only significant P values are shown. (C) Correlation of H5-specific CD4 and CD8 responses with peak neutralization antibody titers (oral in black, n = 10; tonsillar in blue, n = 20; intranasal in red, n = 24) were assessed by the Spearman’s rank method. The XA subgroups were excluded. Only significant P values are shown. In multiple-comparison calculations including all P values, the FDR was estimated to be 6% using the Benjamini-Hochberg procedure, and a fixed P value significance threshold was set at 0.05.

Figure 3. Clinical correlates of anti-H5–neutralizing antibody titers.

(A) The correlation between H5-neutralizing antibody titers 8 weeks (left, n = 32) and 26 weeks (right, n = 33) after vaccination and the last day of viral shedding detected was assessed for participants from the tonsillar (blue squares) and intranasal (red circles) groups who had a 4-fold increase in Ad4-neutralizing antibody titers after vaccination. Significance was determined by Spearman’s rank method. Only significant P values are shown. (B) The correlation between Ad4-neutralizing antibody titers at baseline and the last day of detectable viral shedding (left, n = 48) and peak H5-Vtn neutralization titers (right, n = 49) was assessed by the Spearman’s rank method. Symbols are as in A. Vaccinees who did not seroconvert to Ad4 are included for the purpose of this analysis. (C) The correlations between peak H5-neutralizing antibody titers after vaccination and the duration of related fever (days, left), the total number of participants with related AEs (middle), and the duration of related AEs (days, right) was assessed among vaccine recipients in the oral, tonsillar, and intranasal arms (n = 56; participants with matching AEs and H5-neutralizing antibody titers are shown). The Spearman’s rank method was used to determine significance.

Figure 4. Induction of H5-specific antibodies at mucosal sites.

(A) The proportions of H5 HA-specific IgG (n = 14–37) and IgA (n = 13–37) in nasal (orange), rectal (blue), and cervical (green) secretions were calculated against total IgG and IgA. All available samples from intranasal and tonsillar groups were assessed. Participants who were seropositive for Ad4 at baseline were excluded. Red horizontal bars indicate median values. Generalized estimating equations with an autoregressive correlation structure were used to calculate P values (week 0 measurements were not included), and only significant P values are shown. (B) Shown are serum (n = 15) and cervical secretion (n = 12) concentrations of H5-specific IgG 4 weeks after vaccination and the ratio of H5-specific antibodies in serum versus cervical secretions (n = 12). A paired t test was used to calculate P values. Red horizontal bars indicate median values.

Figure 5. Neutralization antibody titers after a MIV prime/boost.

(A) Immunization schedule. Ad4 (gray triangles), PanBlok (PB) (yellow triangle), and MIV (green triangles) immunizations are shown. (B) Interval between each immunization. Black circles indicate the participants in group 1, and orange circles indicate the participants in group 2 (n = 3). Participants with less than a 4-fold increase in Ad4 neutralization titers after immunization were excluded. (C) Longitudinal changes in the median H5 HA-specific neutralization antibody titers, shown as the median ID50 in serum from participants in group 1 (n = 8), group 2 (n = 3), and group 3 (n = 10), were measured by the PVEI assay. The same exclusion criteria as in B were applied. The intervals between each immunization are shown as disconnected lines between time points. (D) Post-vaccination MIV prime/boost H5 HA-specific neutralizing antibody titers in serum from participants group 1 (black, n = 8) group 2 (orange, n = 3), and group 3 (blue, n = 10) were measured by the PVEI assay. The time points for before and 8 weeks after Ad4 priming (intranasal and tonsillar groups combined are shown in gray; n = 36) are also included for comparison. The same exclusion criteria as in B were applied. Two-sample t tests were used to calculate P values. Red horizontal bars indicate median values. (E) Post-vaccination MIV prime/boost H5 HA-specific neutralizing antibody titers in serum were measured by MN and HAI assays (group 1, n = 8; group 2, n = 3; group 3, n = 10). The same exclusion criteria as in B were applied. Red horizontal bars indicate median values. Two-sample t tests were used to calculate P values. Only significant P values are shown. In multiple-comparison calculations including all P values, the FDR was estimated to be 3% using the Benjamini-Hochberg procedure, and a fixed P value significance threshold was set at 0.05.