Infection of mice with respiratory syncytial virus during neonatal life primes for enhanced antibody and T cell responses on secondary challenge

Abstract

Primary neonatal immune responses to infection or vaccines are weak when compared with those of adults. In addition, memory responses of neonatally primed animals may be absent, weak or T helper type 2 (Th2)-biased. Respiratory syncytial virus (RSV) is an important pathogen of human infants and infection during the neonatal period has been linked to the development of asthma in later life. Here we report that acute intranasal infection of neonatal mice with RSV induces significant RSV-specific antibody and CD8 T cell responses. These responses were boosted after RSV rechallenge during adulthood, demonstrating the establishment of memory after neonatal priming. Primary infection during neonatal life was associated, following rechallenge, with limited viral replication in the lung. Recall responses of both spleen and lymph node cells from neonatally primed and adult-primed mice were associated with interferon-gamma secretion, indicative of a Th1-type response. However, interleukin (IL)-4 and IL-5 secretion were enhanced only in spleen and lymph node cells from neonatally primed mice. Rechallenge of neonatally primed mice was also associated with increased concentrations of chemokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha and regulated upon activation normal T cell expressed and secreted in the lung. These may play a role in the enhanced inflammatory cell recruitment and immunopathology induced following RSV reinfection. Our results demonstrate therefore that immunity to RSV can be established during neonatal life and, importantly, that the quality of the subsequent response is dependent upon the age of first infection.

Fig. 1

Primary respiratory syncytial virus (RSV) infection induces weak antibody responses in neonatal mice, but primes for enhanced responses following subsequent RSV infection. Mice were infected with RSV as adults (adult) or neonates (neonate) and RSV-specific IgM and IgG were quantified by enzyme-linked immunosorbent assay (ELISA) (a). Anti-RSV serum IgG1 and IgG2a responses were measured 21 days after primary adult or neonatal infection (b). Seven weeks after primary infection of adult or neonatal mice, animals were re-challenged with RSV (c). A third group of mice, previously uninfected (naive), were infected similarly with the same dose of RSV. RSV-specific IgG was quantified by ELISA at indicated time-points after infection, and RSV-specific IgG1 and IgG2a were quantified at 21 days after infection. Values represent mean values from a minimum of five mice ± standard deviation. Differences between unprimed adult and neonatally primed groups are shown; *P < 0·05 by Mann–Whitney rank sum test. The data shown are representative of two independent experiments.

Fig. 2

Effective boosting of respiratory syncytial virus (RSV)-specific CD8 T cells in lungs of mice primed as neonates and rechallenged as adults. Analysis of the neonatal primary response is shown in (a); mice were infected with RSV at 7 days of age and at the times indicated post-infection lungs were disrupted and cells analysed for expression of CD8 and RSV peptide-specific T cell receptor and expressed as tetramer-positive cells as a percentage of CD8⁺ cells. Values represent the mean values from at least four mice per group ± standard deviation. Mice primed as adults or neonates were rechallenged with RSV during adult life, 7 weeks post-priming. Mice previously naive were challenged once with RSV. At the times indicated post-secondary challenge with RSV (or primary challenge for the naive group), lungs were disrupted and tetramer and CD8 analysis was performed as above. Fluorescence activated cell sorter plots represent pooled lungs from at least four mice per group, and frequencies of tetramer-positive cells as a percentage of CD8⁺ T cell numbers are shown (b). The data shown are representative of two independent experiments.

Fig. 3

Effective boosting of respiratory syncytial virus (RSV)-specific interferon (IFN)-γ-secreting CD8 T cells in lungs and spleens of mice primed as neonates. Analysis of the neonatal primary response in the lung (a) and spleen (b) is shown. Mice primed as adults or neonates were rechallenged with RSV during adult life 7 weeks post-priming (c and d). Mice previously naive were challenged once with RSV. At the times indicated post-secondary challenge with RSV (or primary challenge for the naive group, and neonatal primary response), RSV peptide-specific IFN-γ-secreting cells in lung (a and c) and spleen (b and d) were detected by enzyme-linked immunospot assay (ELISPOT). Values represent means of at least five mice ± standard deviation. Insignificant differences between adult-primed and neonatally primed groups are shown for day 4; *P = 0·314 (lung) and **P = 0·280 (spleen) by Student's t-test. The data shown are representative of two independent experiments.

Fig. 4

High-level interleukin (IL)-5 production is evident only in neonatally primed lymph node and spleen following respiratory syncytial virus (RSV) infection during adult life. Mice primed as adults or neonates were rechallenged with RSV during adult life 7 weeks post-priming. Mice previously naive were challenged once with RSV. Spleens and lymph node were harvested 7 days after secondary challenge (or primary challenge for the naive group). Spleen or lymph node cells were then restimulated in culture with RSV-infected syngeneic, irradiated splenocytes (a) or with medium alone (b) for 72 h. The IL-5 and interferon-γ content of cell culture supernatants (pg/ml) was assessed. IL-4 secreting cells were detected by enzyme-linked immunospot assay (ELISPOT) and expressed as spots/10⁶ cells (c). Results for individual mice are shown for spleen cell cultures, and for pooled lymph node cultures (from at least four mice per group) and each bar represents the average ± standard deviation values from triplicate enzyme-linked immunosorbent assay wells. Differences between adult- and neonatally primed groups are shown; *P < 0·05 and **P < 0·01 and ***P < 0·001 by Student's t-test. The data shown are representative of two independent experiments.

Fig. 5

Neonatal infection with respiratory syncytial virus (RSV) protects against secondary challenge. Mice primed as adults or neonates were rechallenged with RSV during adult life, 7 weeks post-priming. Mice previously naive were challenged once with RSV. Lungs were harvested 4 days after secondary challenge (or primary challenge for naïve group) and viral titre determined by plaque assay and expressed as plaques per gram of lung. Data are expressed for individual mice and means shown.

Fig. 6

High level of inflammatory cytokines and chemokines in lungs of neonatally primed mice following secondary challenge with respiratory syncytial virus (RSV) during adult life. Mice primed as adults or neonates were rechallenged with RSV during adult life 7 weeks post-priming. Mice previously naive were challenged once with RSV. Lungs were harvested 4 days after secondary challenge (or primary challenge for the naive group) and concentration of cytokines and chemokines in supernatants were determined by cytometric bead array assay or enzyme-linked immunosorbent assay and expressed as pg/ml of supernatant. Differences between adult- and neonatally primed groups are shown; *P < 0·05, **P < 0·01 and ***P < 0·001 by Student's t-test. Data are expressed for individual mice. The data shown are representative of two independent experiments.