Impaired Antibody-mediated Protection and Defective IgA B-Cell Memory in Experimental Infection of Adults with Respiratory Syncytial Virus

Abstract

Rationale: Despite relative antigenic stability, respiratory syncytial virus (RSV) reinfects throughout life. After more than 40 years of research, no effective human vaccine exists and correlates of protection remain poorly defined. Most current vaccine candidates seek to induce high levels of RSV-specific serum neutralizing antibodies, which are associated with reduced RSV-related hospitalization rates in observational studies but may not actually prevent infection.

Objectives: To characterize correlates of protection from infection and the generation of RSV-specific humoral memory to promote effective vaccine development.

Methods: We inoculated 61 healthy adults with live RSV and studied protection from infection by serum and mucosal antibody. We analyzed RSV-specific peripheral blood plasmablast and memory B-cell frequencies and antibody longevity.

Measurements and main results: Despite moderately high levels of preexisting serum antibody, 34 (56%) became infected, of whom 23 (68%) developed symptomatic colds. Prior RSV-specific nasal IgA correlated significantly more strongly with protection from polymerase chain reaction-confirmed infection than serum neutralizing antibody. Increases in virus-specific antibody titers were variable and transient in infected subjects but correlated with plasmablasts that peaked around Day 10. During convalescence, only IgG (and no IgA) RSV-specific memory B cells were detectable in peripheral blood. This contrasted with natural influenza infection, in which virus-specific IgA memory B cells were readily recovered.

Conclusions: This observed specific defect in IgA memory may partly explain the ability of RSV to cause recurrent symptomatic infections. If so, vaccines able to induce durable RSV-specific IgA responses may be more protective than those generating systemic antibody alone.

Keywords: antibody-secreting cells; immunologic memory; mucosal immunity; nontherapeutic human experimentation; respiratory syncytial virus vaccines.

Figure 1.

Respiratory syncytial virus (RSV)–specific nasal IgA is a superior correlate of protection to serum neutralization titer. (A) Serum neutralizing antibody (Ab) was determined by plaque reduction neutralization and nasal IgA by ELISA. Baseline serum neutralizing (top), nasal IgA against whole RSV (middle), and nasal anti-RSV fusion F protein (bottom) Ab levels in uninfected (open circles) versus infected (solid circles) subjects are shown. Serum neutralization includes four infant samples (open triangles) and three reference serum standards (solid squares; Wyeth 06937, 06938, and 06939). Horizontal bars indicate the median. (B) Logistic regression of probability of protection with baseline serum neutralizing (top), nasal whole RSV IgA (middle), and nasal RSV fusion F protein IgA (bottom) Ab levels with predicted probability (solid line) and point-wise 95% confidence band (shaded) are shown. EC₅₀ = half-maximal effective concentration; OR = odds ratio (and 95% confidence interval). P values for unpaired Mann-Whitney Wilcoxon U test or two-tailed z test as appropriate are shown. ns = P > 0.05; *P < 0.05; **P < 0.01.

Figure 2.

Serum and nasal antibody (Ab) increase after infection but are not maintained. Healthy adult volunteers were challenged intranasally with respiratory syncytial virus (RSV) A M37. Serum and nasal lavage was taken at baseline and 28 days postinoculation. A subset of subjects returned for further blood and nasal sampling 6–12 months later (nominally Day +180). Serum neutralizing Ab was determined by plaque reduction neutralization and nasal IgA by ELISA. (A) Fold change versus baseline of serum in serum plaque reduction neutralization titer (top), nasal IgA against whole RSV (middle), and nasal anti-RSV fusion F protein (bottom) Ab levels in uninfected (open circle) versus infected (solid circle) subjects. Horizontal bars indicate the median. (B) Individual plots showing trend in Ab levels at baseline and Days +28 and +180. P values for unpaired Mann-Whitney Wilcoxon U test are shown. EC₅₀ = half-maximal effective concentration. ***P < 0.001.

Figure 3.

Respiratory syncytial virus (RSV)–induced plasmablast expansion correlates with IgG and IgA increment. (A) Antigen-specific antibody (Ab)-secreting cells (ASCs) were enumerated using B-cell enzyme-linked immunospot with whole RSV lysate where sufficient peripheral blood mononuclear cells (PBMCs) were available. Uninfected (polymerase chain reaction–negative) subjects (n = 12) are shown in blue, infected (polymerase chain reaction–positive) subjects (n = 16) in red. (B) Peak antigen-specific ASC frequencies as percentage of total immunoglobulin-positive cells for infected subjects. Horizontal bars indicate the median. (C) Correlation of fold change in serum neutralizing Ab with peak IgG⁺ ASC frequency. (D) Correlation of fold change in nasal IgA against RSV with peak IgA⁺ ASC frequency (data from 27 subjects; nasal IgA in one subject could not be measured). (E) Correlation of peak ASC frequency with preinfection baseline serum neutralizing Ab titers. ASC frequencies are expressed as log₁₀ per million PBMC. P values for unpaired Mann-Whitney Wilcoxon U tests and/or Spearman rank correlation coefficient (R_s) are shown as appropriate. Open circles = uninfected subjects; solid circles = infected subjects. Ab = antibody; EC₅₀ = half-maximal effective concentration. ns = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4.

Antigen-specific IgG⁺ memory B cells (MBC) are induced by respiratory syncytial virus (RSV) infection. Peripheral blood mononuclear cells (PBMCs) were obtained on Days 0 and 28 postinoculation and MBCs polyclonally activated. Total and antigen-specific immunoglobulin-producing cells were then enumerated using enzyme-linked immunospot. (A) Antigen-specific IgG⁺ MBC frequencies for uninfected (open circles) versus infected (solid circles) subjects at baseline and Day 28 postinoculation (infected only). P > 0.05 for all comparisons except where shown. MBC frequencies are expressed as percentage of total immunoglobulin-positive cells. Horizontal bars indicate the median. (B) Fold change (log₂) of IgG⁺ MBCs postinfection compared with baseline for infected subjects (n = 17). Horizontal bars indicate the median. (C) Correlation between peak acute antibody-secreting cell (ASC) frequency at Day 10 and fold change over baseline (log₂) of RSV-specific IgG⁺ MBC. Spearman rank correlation coefficient (R_s) or P values for unpaired Mann-Whitney Wilcoxon U test are shown as appropriate. FP = purified RSV fusion protein; MV = measles virus; RSV = whole RSV. ns = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.

Respiratory syncytial virus (RSV) infection fails to induce IgA⁺ memory B cells (MBCs). Peripheral blood mononuclear cells were obtained on Days 0 and 28 postinoculation and MBCs activated polyclonally in vitro. Total and antigen-specific immunoglobulin-producing cells were then enumerated using B-cell enzyme-linked immunospot as before. Data for infected subjects exhibiting adequate peripheral blood mononuclear cell responses to in vitro stimulation only (n = 17) are shown. (A) Frequency of IgA⁺ MBCs at baseline (preinfection) and (B) convalescence (Day +28). Subjects experimentally challenged with RSV A M37 were compared with patients naturally infected with pandemic influenza A/H1N1/09 (seven exhibited adequate response to stimulation and were analyzed). (C) IgG⁺ MBC frequencies and (D) IgA⁺ MBC frequencies at Day 28 postinoculation (RSV) and Day 28–42 posthospitalization (H1N1) are shown. Horizontal bars indicate the median. P values for unpaired Mann-Whitney Wilcoxon U test are shown as appropriate. FP = purified RSV fusion protein; MV = measles virus; RSV = whole RSV. ns = P > 0.05; **P < 0.01.