Immune responses following neonatal DNA vaccination are long-lived, abundant, and qualitatively similar to those induced by conventional immunization

Abstract

Virus infections are devastating to neonates, and the induction of active antiviral immunity in this age group is an important goal. Here, we show that a single neonatal DNA vaccination induces cellular and humoral immune responses which are maintained for a significant part of the animal's life span. We employ a sensitive technique which permits the first demonstration and quantitation, directly ex vivo, of virus-specific CD8(+) T cells induced by DNA immunization. One year postvaccination, antigen-specific CD8(+) T cells were readily detectable and constituted 0.5 to 1% of all CD8(+) T cells. By several criteria-including cytokine production, perforin content, development of lytic ability, and protective capacity-DNA vaccine-induced CD8(+) memory T cells were indistinguishable from memory cells induced by immunization with a conventional (live-virus) vaccine. Analyses of long-term humoral immune responses revealed that, in contrast to the strong immunoglobulin G2a (IgG2a) skewing of the humoral response seen after conventional vaccination, IgG1 and IgG2a levels were similar in DNA-vaccinated neonatal and adult animals, indicating a balanced T helper response. Collectively, these results show that a single DNA vaccination within hours or days of birth can induce long-lasting CD8(+) T- and B-cell responses; there is no need for secondary immunization (boosting). Furthermore, the observed immune responses induced in neonates and in adults are indistinguishable by several criteria, including protection against virus challenge.

FIG. 1

Virus-specific CD8⁺ T cells are detectable directly ex vivo 1 year after DNA immunization of neonates or adults. Neonatal or adult mice were immunized with pCMVNP or pCMV. One year later, splenocytes were assayed, directly ex vivo, by flow cytometry for expression of CD8 (x axis) and IFN-γ (y axis) after 5 h of peptide stimulation as described in Materials and Methods. As positive controls, splenocytes were included from LCMV-infected mice at either 7 (d7) or 246 (d246) days p.i. The number in the top right-hand corner of each dot plot indicates the percentage of CD8⁺ cells which were IFN-γ⁺ (i.e., specific for the CTL epitope peptide NP_118–126). The data shown are representative of three independent experiments.

FIG. 2

Perforin is detectable in CD8⁺ memory T cells induced by DNA vaccination or virus infection. CD8⁺ T cells from the same splenocytes used in the experiment shown in Fig. 1 were stained for perforin and IFN-γ and analyzed by flow cytometry. For all mice, perforin-specific fluorescence was measured in virus-specific CD8⁺ T cells (i.e., those which produced IFN-γ in response to peptide) and also in nonresponding (IFN-γ⁻) CD8⁺ T cells. A representative mouse from each group is shown (solid line), and the median fluorescence is indicated in the upper right corner of each histogram. Splenocytes from perforin-deficient mice were included as a negative staining control and are shown as a dotted line in each histogram; the median fluorescence of perforin-deficient CD8⁺ T cells was 12.9. d7, day 7; d246, day 246.

FIG. 3

Accelerated antigen-specific lytic CD8⁺ T cell responses at 6 months and 1 year after DNA vaccination of neonatal or adult mice. Adult (▾, panels A and C) or neonatal (●, panels B and D) mice received a single 50-μg injection of pCMVNP. Six months (panels A and B) or 1 year (panels B and D) later, mice were infected with LCMV, and 4 days thereafter were evaluated for the presence of lytic CTL activity. Each line represents the percent specific ⁵¹Cr release for an individual mouse, determined at the indicated effector-to-target cell (E:T) ratios. For each experiment, the nonimmune mouse (either pCMV inoculated or given no DNA) showing the highest level of background lysis at day 4 p.i. is shown (open squares).

FIG. 4

DNA vaccination of neonates or adults induces long-lived IgG responses. Neonatal mice (circles) or adult mice (triangles) were injected with pCMV (open symbols) or pCMVNP (solid symbols). The representative experiment shown used four mice per vaccine group. Six months later, serum from each mouse was tested by enzyme-linked immunosorbent assay for the presence of LCMV-specific antibodies as described in Materials and Methods. At each serum dilution, the mean and standard error for each vaccine group are shown.

FIG. 5

Similar isotype responses in mice immunized as neonates or adults. The isotype profile of the LCMV-specific antibodies induced by pCMVNP immunization of adult (A) or neonatal (B) mice are shown. Black bars, IgG1; gray bars, IgG2a. Each paired set of bars shows the average titers for a single animal, and error bars representing the standard deviation are included. For comparative purposes, the isotype profile present 6 weeks after virus infection is shown (C). Note that the left-hand y axis scale refers to panels A and B, and the right-hand y axis scale refers to panel C.

FIG. 6

Neonatal and adult DNA immunization provides long-term protection against viral challenge. Neonatal and adult mice were immunized as indicated. Six months (A) or 1 year (B) postimmunization, the mice were challenged with LCMV, and 4 days later their spleens were harvested for virus titration. Each bar represents the amount of infectious virus present per gram of spleen in a single animal at 4 days p.i. (d4) (or 7 days p.i. for the d7 control mice). To display the correlation between protective immunity and the presence of CTL, the bars are color coded; white bars designate animals in which lytic CTL activity was detected in the experiment shown in Fig. 3, while black bars designate animals devoid of CTL activity. The two virus control groups shown in Fig. 3 were also analyzed. The virus titers in nonimmune mice infected with LCMV 4 days prior to titration (d4) were used as a baseline against which the DNA vaccines were compared. The horizontal bars indicate a 99.9% (1,000-fold) decrease from the average titers of these d4 mice. The lower limit of detection is 200 PFU per gram; mice in which plaques were undetectable (but which displayed CTL activity and thus had been successfully infected) are indicated by asterisks.