Relation between Ab specificity, titer and protection
We found that the concentration of M2e(pep-nat)-specific Abs in sera of parenterally vaccinated mice correlated with strength of protection (Table
1). This is consistent with previous studies showing that protection can be transferred to naive mice by passive M2e-specific Abs [
10,
11,
17] and antisera [
4‐
6,
8,
9]. It is consistent also with the generally held view that M2e-specific Abs mediate protection by reaction with M2e expressed in the plasma membrane of infected host cells. By contrast, M2e(pep)-specific Ab titers showed no correlation with protection, in spite of the fact that the M2e(pep-nat)-specific Abs are a fraction of the larger M2e(pep)-specific Ab response (Table
1). This lack of correlation between M2e(pep) Ab titer and protection appears to be a consequence of the large variability of the M2e(pep-nat)-fraction within the M2e(pep) response (Fig
2C). The reason for this variability is not known. It could be due to a low frequency of M2e(pep)- and M2e(pep-nat)-specific B cells within the naive B cell repertoire, which may result, for stochastic reasons, in large differences in the composition of this response between individuals and even pooled sera from 3–5 animals, as tested here. A low precursor B cell frequency is consistent with our previous finding that seven M2e(pep-nat)-specific hybridomas isolated from three mice all expressed a highly restricted heavy chain variable region (formed by recombination of the same V
H, D and J gene segments) in association with only two distinct V
L genes [
23]. The M2e(pep)-specific response has not been analyzed at the clonal level but may be equally restricted. Because of this variability, the protective M2e(pep-nat)-specific Ab titers cannot be extrapolated from M2e(pep)-specific Ab titers and must be measured specifically.
Given the importance of M2e(pep-nat)-specific Abs in protection, the selective promotion of this Ab population by a M2e vaccine would be advantageous. This may be achieved by development of a more effective vaccine construct and/or vaccine administration. Of note in the latter context is the present finding that concomitant administration of M2e-MAP and a sublethal dose of infectious virus by the i.n. route not only enhanced the M2e(pep-nat)-specific serum Ab titer compared to vaccination with infectious virus or M2e-MAP (plus adjuvants) alone, but affected also the specificity of the response in that essentially all M2e-specific Abs generated in these co-immunized mice displayed M2e(pep-nat)-specificity (Fig
7A and data not shown). The advantage of co-administration of infectious virus and M2e-MAP with regard to strength of protection against heterosubtypic IAV challenge (as used in the present study) merit further investigation, particularly since this protocol may be adaptable to humans in the form of i.n. vaccination with a combination of live attenuated IAV and a M2e-vaccine.
The relation between M2e(pep-nat)-specific Ab titers in sera of parenterally vaccinated mice and strength of protection followed sigmoidal curves (Fig
6A), which suggested that M2e(pep-nat)-specific serum Abs were equally protective in nose, trachea and lung (EC
50~20 μg/ml). This was unexpected in view of previous studies showing that systemically administered passive anti-viral Abs of IgG isotypes were significantly less protective in upper than lower airways [
24‐
26]. The reason for this appears to be the lower rate of transudation of serum IgG through the pseudostratified columnar epithelium of upper airways than the thinner epithelium of respiratory airways and alveoli [
27]. To confirm that this differential effectiveness applies also to M2e(pep-nat)-specific Abs, we injected fifteen naive BALB/c mice with three different purified mAbs (5 mice/Ab) to achieve a passive serum Ab concentration of ~20 μg/ml and then challenged the mice by i.n. inoculation of 5 μl X31. Determination of virus titers in lung, trachea and nose five days later confirmed the decreasing protective activity of serum Ab from lower to upper airways, in that mAb-treated mice exhibited, on average, a 100 fold reduction in virus titer in the lung, 30 fold in the trachea and no reduction at all in the nose compared to control mice treated with PBS (data not shown). Accordingly, M2e(pep-nat)-specific serum Ab titers in mice that had been immunized by a parenteral route appeared to account reasonably well for the protection in lung and trachea but not in the nose.
One possible explanation for this difference in protection between actively and passively immunized mice was that active immunization induced substantial levels of M2e(pep-nat)-specific IgA. When dimerized with J chain, IgA is actively transported by the polymeric Ig receptor (pIgR) system through the columnar epithelium of conducting airways and is therefore more abundant than IgG in secretions of upper than lower airways [
27,
28]. Accordingly, secretory IgA with virus-neutralizing activity has been shown to be responsible for much of the protection against IAV replication in the nasal cavity of mice, while IgG is more important for protection of respiratory airways [
29‐
31]. However, we could not detect significant levels of M2e(pep-nat)-specific IgA in sera of parenterally vaccinated mice (data not shown), making this explanation untenable. Another possibility, which is discussed in more detail below, is that parenteral vaccination with M2e-MAP induced significant airway-associated immunity. Although induction of strong local airway-associated immunity is generally thought to require administration of antigen into the airways [
31‐
33], there is evidence indicating that parenteral immunization with CT may result in the migration of dendritic cells to mucosa-associated lymphoid tissues and thereby promote some level of mucosa-associated immunity [
34,
35]. In this study, CT significantly enhanced the systemic Ab response upon parenteral vaccination but we do not know whether it also resulted in the induction of nasal mucosa-associated immunity that may have restricted virus replication in nasal tissue, independent of serum Ab titer. Finally and probably most likely, immunization with M2e-MAP may have induced not only M2e-specific Abs but also T cells that contributed to protection. This possibility is supported by previous studies showing that vaccination of BALB/c mice with M2e-MAP [
17] or M2-DNA and M2-recombinant adenovirus [
9] induced M2e-specific T cell responses, most likely of CD4 phenotype, and that virus-specific CD4 memory T cells could significantly restrict virus replication in the nose but not the lung [
36]. Accordingly, M2e-specific CD4 T cells may have inhibited virus replication in the nose and M2e-specific serum Abs in the lung. This proposition does not conflict with the conclusion of Jegerlehner et al. [
6] that M2e-specific T cells played no role in protection of mice against a lethal total respiratory virus challenge, as the lethality of the infection is determined by the level of virus replication in the lung but not the nose. The contrasting finding by Tompkins et al. [
9] that T cells contributed to protection against lethal IAV challenge in mice immunized by M2-DNA and M2-recombinant adenovirus may be explained by induction of M2-specific CD8 T cells in these mice. It is well established that virus-specific memory CD8 T cells can contribute to resistance against a lethal IAV challenge.
Route of vaccination and strength of protection
I.n. vaccination resulted in stronger protection against descending infection than parenteral vaccination (Fig
3C,D). Most remarkably, however, the strength of protection in i.n. vaccinated mice showed no correlation with M2e(pep-nat)-specific serum Ab titers (Table
1). Indeed, several groups of i.n. vaccinated mice with serum Ab titers that were completely non-protective in parenterally vaccinated mice showed nevertheless strong protection (Fig
6B,C). Several explanations can be considered.
First, i.n. administration of adjuvant alone has been shown to result in a temporary increase in resistance against virus replication in the respiratory tract [
37‐
40]. However, such a non-specific enhancement of resistance is unlikely to have affected the results of this study, since M2e-MAP-vaccinated mice were always compared to control mice that had been vaccinated by the i.n. route with adjuvant alone, thus canceling out adjuvant-induced non-specific effects.
Second, i.n. vaccination may have induced local, airway-associated immunity that was not adequately reflected by serum Ab titers. To affect virus replication, Abs must be present in airway secretions. Abs in this location may have two distinct provenances [
41]: 1) They may be serum Abs that transudated into extravascular spaces of airway tissues and, in the case of IgG, transudated further into the airway lumen or, in the case of IgA and IgM, became transported through the epithelial cell layer by pIgR. 2) They may have been secreted by B cells located in the lamina propria of airways. As such locally produced Abs, particularly J-chain associated IgA and IgM, can be expected to be delivered more effectively into the airway lumen than into the intravascular compartment, serum Ab titers do not provide a reliable measure of the locally produced fraction of Abs. The importance of nasal administration of vaccine for promotion of local immunity has been documented both in animal models [
31,
42‐
45] and humans [
46‐
49]. Once induced, antigen-specific B and T cells may persist in airway tissues for an extended period of time and provide the host with long lasting enhanced protection [
50‐
54]. Accordingly, local M2e(pep-nat)-specific B and possibly also T cells may have provided strong protection in some i.n. vaccinated mice in the absence of protective serum Ab titers (Fig
6B,C).
Third, i.n. vaccination may have induced a qualitatively different and more protective immune response than parenteral vaccination. It is well established, for instance, that i.n. vaccination typically promotes a stronger IgA response than parenteral vaccination. The fact that we could not detect significant M2e(pep-nat)-specific IgA in pooled sera of i.n. vaccinated mice (data not shown) does not exclude the possibility that M2e(pep-nat)-specific IgA was produced locally and efficiently transported into airway secretions. In contrast to IgG, locally produced IgA may interact intracellularly with M2e during its pIgR-mediated transport through infected epithelial cells and thereby restrict virus replication [
55]. The substantial efficacy of this mechanism in vivo has been demonstrated by passive IgA mAb-mediated clearance of Rotavirus from intestinal epithelium of mice with severe combined immunodeficiency [
56]. After its release into airway secretions, secretory M2e(pep-nat)-specific IgA may have lesser protective power than IgG, both in terms of activation of FcR-expressing effector cells and complement. Nevertheless, cell-bound secretory IgA, while incapable of activating effector cells through one of the widely expressed activating FcγRs, may still be able to activate effector cells through interaction with the recently identified FcαμR in mice [
57] or CD86 in humans. In addition, while incapable of activating complement through the classic pathway, IgA may still activate it through the alternative [
58] and lectin [
59] pathways if complement activation were involved in M2e-Ab-mediated protection. Another potentially important qualitative change observed here after i.n. administration of vaccine was the significant increase in the proportion of M2e(pep-nat)-specific Abs of G2a isotype (Fig
4B). Firstly, IgG2a was the most protective IgG isotype in passive transfer experiments (Fig
5). In addition, if T cells contributed to protection, the prevalence of IgG2a may indicate a general bias of the response towards type 1, which is typically associated with optimal T cell-mediated protection in viral and bacterial infections. Additional studies are needed to sort out the relative importance of local immunity and quality of the response in the improved protection after i.n. vaccination.
The enhanced protection seen here after i.n. vaccination must be viewed in the context of the challenge used here. It consisted of an infection that was initially confined to the nasal epithelium and allowed to descend from there into the lower respiratory tract over the course of five days. In this scenario, strong immunity in the upper respiratory tract would be expected to have a substantial impact on the progress of the infection. By contrast, the more frequently used challenge with an inoculum of 30–50 μl in anesthetized mice initiates an infection in both upper and lower respiratory tact, and virus titer in lung or survival would hardly if at all be affected by immunity in the upper respiratory tract. We believe this nasal challenge provides a relevant model for the IAV infection in humans.