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*
Department of Pediatrics, Vanderbilt University, Nashville, TN 37232; and
Respiratory Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinical and laboratory studies provide strong evidence that Abs, including mucosal and serum Abs, play a dominant role in protection against infection or reinfection by respiratory viruses (5, 6). The relative sparing of infants from virus-associated lower respiratory tract disease in the first weeks or months of life correlates with the level of passively acquired maternal Abs to RSV (7), parainfluenza virus type 3 (PIV3) (8), or influenza viruses (9). Administration of pooled human IgG containing a high titer of RSV neutralizing Abs or of a humanized murine RSV-specific mAb prevents severe RSV disease in high-risk infants and children (10, 11). Passive transfer of RSV polyclonal immune sera or neutralizing RSV mAbs protects the lower, and to a lesser extent, the upper respiratory tract of rodents against replication of RSV challenge virus (12, 13, 14, 15). The F (fusion) and G (attachment) glycoproteins are the major protective Ags of RSV, and only these RSV Ags induce neutralizing Abs (2). A major goal of active RSV immunization is to induce high levels of serum and mucosal neutralizing Abs that are able to protect the upper (16, 17) and lower respiratory tract against a high level of replication of wild-type (wt) RSV.
Unfortunately, infants in the first 6 mo of life mount a poor primary
Ab response to respiratory virus infection or immunization (5, 6). The overall level of serum and mucosal Ab response to the F
and G glycoproteins of young infants to primary RSV infection is
15–25% that of older infants and children (18, 19).
Two principal factors contribute to this diminished response, namely,
maternal Ab-mediated immune suppression and immunological immaturity
(18). A high level of maternally acquired RSV Abs
decreased the serum Ab response to the G glycoprotein, and young age
diminished the response to the F glycoprotein in infants with RSV
infection (18). In experimental animals, passive transfer
of RSV Abs decreased the frequency and magnitude of the Ab response to
F and G proteins expressed by vaccinia virus recombinants and decreased
the neutralizing activity of the Ab that was induced (20).
In seronegative chimpanzees, passive RSV Abs suppressed the primary Ab
response to infection with live attenuated RSV virus vaccine candidates
but, unexpectedly, did not inhibit the development of resistance to wt
virus challenge or the secondary Ab response to infection with RSV
challenge virus (21). The mechanisms underlying the
protective efficacy of live attenuated vaccines in chimpanzees
immunized in the presence of passively acquired RSV Abs have not been
defined. The diminished Ab response to immunization suggested that
other immune mediators, such as T cells, might contribute to the
observed resistance to replication of RSV challenge virus.
In the present study, the immunogenicity and efficacy of two live attenuated RSV vaccine candidates differing in their level of attenuation were evaluated in mice passively immunized with varying quantities of RSV Abs. The live RSV vaccine candidates induced a high level of immunity in mice, as in chimpanzees (21), when immunization was performed in the presence of physiological levels of passively transferred RSV neutralizing Abs. Immunity exhibited by these mice was shown in the present study to be mediated by CD4+ and CD8+ T cells as well as by Abs. CD4+ and CD8+ T cells made a greater contribution in those mice with a high level of Ab-mediated suppression of the humoral immune response, whereas humoral immunity played the major role in those mice with a strong humoral immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HEp-2 cells, obtained from the American Type Culture Collection (Manassas, VA) at passage 364, were maintained as previously described (22) and were not used beyond passage 400. HEp-2 cell monolayer cultures grown on 24-well tissue culture plates (Costar) were used for all virus assays as previously described (22).
Viruses
The wt RSV strain A2 and the isolation and characterization of the cold-passaged temperature-sensitive (cpts)-248 and cpts-248/804 mutants were described previously (23, 24, 25). Briefly, the wt RSV strain A2 was multiply passaged at low temperature to yield a host-range mutant, designated cp-RSV, that was subsequently chemically mutagenized to yield a temperature-sensitive (ts) mutant designated cpts-248. The shut-off temperature of cpts-248 (lowest temperature at which a 100-fold reduction in plaque titer vs titer at permissive temperature occurs) was 38°C. The cpts-248 mutant was then mutagenized to yield a ts mutant with a lower shutoff temperature, cpts-248/804 (37°C shutoff). Each of the viruses was biologically cloned by three plaque-to-plaque passages in Vero cells and subsequently amplified in Vero cell monolayer culture. These mutants differed in their replicative capacity in rodents or chimpanzees, with cpts-248 replicating to a high level, whereas cpts-248/804 replicated to an intermediate level (24, 25). Each virus was concentrated by ultracentrifugation as previously described (25) to achieve the high virus input titer needed in experiments performed in mice. A similarly prepared wt PIV3 (strain JS) suspension was used as a heterologous virus control in some experiments.
Virus titrations
Virus present in tissue culture harvests or in the lung or nasal turbinate (NT) homogenates of mice was quantitated by plaque assay on HEp-2 cell monolayer cultures maintained under a semisolid methylcellulose overlay at 37°C as described, except that plaque assays of the ts mutants were performed at the permissive temperature of 32°C (22). Staining monolayers with RSV-specific murine mAbs using an immunoperoxidase procedure (26) facilitated detection of plaques.
Immunological studies
Animals. Respiratory-pathogen-free BALB/c mice were obtained from Charles River Breeding Laboratories (Wilmington, MA) and were studied at 24–32 wk of age. Mice within each experiment were age-matched. Animals were maintained in microisolator cages throughout the studies. All experiments involving animals were reviewed and approved by the National Institutes of Health Animal Care and Use Committee.
Serology. Serum IgG Abs binding to RSV F surface glycoprotein were quantitated in an ELISA using F glycoprotein that had been immunoaffinity-purified from RSV subgroup A (Long strain)-infected cell lysates as described (19, 27). The neutralizing Ab titer of serum samples was determined by a complement-enhanced 60% plaque reduction assay in HEp-2 cells (28); plaques were visualized using the RSV-specific immunohistological staining procedure (26). Neutralizing Ab titers were expressed as the reciprocal of the highest Ab (or serum) dilutions that yielded a 60% reduction in PFU as compared with the control wells.
ELISPOT assay. Murine Ab-forming cells (AFC) secreting RSV-F-specific Abs of the IgM, IgA, or IgG isotypes were quantitated in an ELISPOT assay. Flat-bottom Immulon I immunoassay plates (Nunc, Naperville, IL) were coated overnight with 200 ng/well immunoaffinity-purified RSV F glycoprotein in carbonate buffer, pH 9. Plates were washed with buffer, and serial 2-fold dilutions of harvested cell populations in RPMI 1640 medium with 2.5% FBS were inoculated in triplicate onto plates and incubated in 5% CO2 at 37°C for 6 h. Cell suspensions were removed, then plates were blocked with PBS/1% BSA. RSV F protein-specific Abs secreted by the cells were detected as spots using peroxidase-labeled isotype-specific goat anti-mouse Abs (Southern Biotechnology Associates, Birmingham, AL) at a 1/1000 dilution in PBS/1% BSA, incubated overnight, then washed three times with PBS/0.05% Tween 20 and deionized water, and overlaid with 50 µl/well substrate (a 1:1 mixture of 2% low-melting-point agarose and NBT/BCIP substrate solution; Sigma, St. Louis, MO). Spots were enumerated under a dissecting microscope, and expressed as number of AFC per 106 total cells.
Preparation of RSV immune and nonimmune serum. Twenty-four-week-old BALB/c mice were infected by the intranasal (i.n.) route with 106.3 PFU of the wt RSV strain A2 virus on two occasions 1 mo apart, then bled for serum collection once a week for 4 wk, beginning 2 wk after the second administration of virus. Sera were pooled and heat-inactivated at 56°C for 30 min. The serum pool had a neutralizing Ab titer of 1:20,000. This serum pool was diluted 1/5 in sterile PBS to generate the working suspension for passive immunization designated "mouse immune serum" (MIS). Control BALB/c mice of an identical age that were not infected with RSV were bled concomitantly to generate a nonimmune serum pool that had a RSV neutralizing Ab titer of <1:20. This serum pool was likewise heat-inactivated and diluted 1/5 with PBS to generate the working suspension designated "nonimmune mouse serum" (NMS).
Passive immunization. Mice were inoculated by the i.p. route with 1 ml of MIS, with MIS further diluted 1/10 or 1/100 with PBS, with NMS, or with PBS alone on the day before primary i.n. immunization with virus. Data from groups treated with MIS 1/10 and MIS 1/100 were similar, and data from the groups treated with NMS or PBS were similar; therefore, only the data from MIS 1/10 and NMS are shown.
Active immunizations. Mice were immunized by the i.n. route on day 0 with 106.5 PFU of virus in a 0.1-ml inoculum. The viruses used were RSV cpts-248, cpts-248/804, wt RSV strain A2, or a heterologous control virus (wt PIV3 strain JS) as indicated in Tables I-V. On day 4 following inoculation, a subset of mice from each treatment group was sacrificed by CO2 inhalation, and NT and lung tissues were harvested for quantitation of virus and characterization of recovered virus as described (24). Twenty-eight days following the initial virus infection, mice were challenged i.n. under anesthesia with 106.3 PFU of wt RSV strain A2 in a 0.1-ml inoculum. On day 4 following challenge, a subset of three mice from each treatment group was sacrificed, and NT and lung tissues were obtained for virus titration. Groups of six mice were bled postinfusion (day 0), postimmunization (day 28), and postchallenge (day 56) to obtain serum for assay of neutralizing or ELISA Ab titer.
Regional Ab and AFC samples. Subsets of animals from each treatment group were sacrificed by CO2 inhalation, and serum, bronchoalveolar lavage (BAL) fluid, and nasal wash (NW) fluid were collected on day 28 (before challenge), or on days 32, 35, or 56 (days 4, 7, or 28 after wt virus challenge) to determine the level of RSV-specific Abs present. Blood was obtained by heart puncture. BAL fluids were obtained by ligation of the trachea with suture, insertion of a 23-gauge blunt needle into the distal trachea, followed by three in-and-out flushes of the airways with 3 ml of sterile PBS. NW were obtained by flushing 3 ml of PBS through the upper trachea and out the nasal orifice into a sterile receptacle. Both BAL and NW were concentrated 10-fold using 50 kDa molecular mass cutoff spin concentrators (Millipore, Bedford, MA).
Lymphocyte populations were harvested from lymphoid tissues on days 28, 32, or 35 for quantitation of RSV-specific AFC using the ELISPOT technique. Cells were pooled from groups of five (day 28) or three animals (days 32 and 35). Total cells were obtained from cervical lymph nodes (CLN), mediastinal lymph nodes (MLN), or spleen by passage of the entire organ through a sterile wire mesh screen into RPMI 1640 with 5% FCS, 50 µg/ml gentamicin, and 2.5 µg/ml amphotericin (cRPMI). Nasal associated lymphoid tissue (NALT) was obtained by dividing the soft and hard palates through the midline, then gently scraping all visible soft tissue into cRPMI, followed by passage through a wire mesh screen. Mononuclear cell fractions were obtained from the lungs as follows. The pulmonary vasculature was cleared of blood by in situ flushing with 3–5 ml of cold sterile PBS administered via 23-gauge needle inserted into the right ventricle. The complete lungs were removed and immersed in cRPMI containing DNase I (Sigma) and collagenase type III (Worthington Biochemical, Lakewood, NJ), minced into fine pieces with surgical scissors, then shaken gently at 37°C for 1 h. The resulting tissue was passed through a wire mesh screen, then the single cell suspension was layered onto a density gradient (Lympholyte M; CEDARLANE Laboratories, Hornby, Ontario, Canada), and centrifuged at 400 x g for 20 min. The mononuclear cell fraction obtained from the gradient interface was resuspended and washed three times in cRPMI. The cell count of the final suspensions of NALT, CLN, MLN, lung cells, or spleen cells was determined using a microscope and hemacytometer. The resulting cell suspensions were used in the ELISPOT assay.
T cell depletion studies
Lymphocyte-depleting or isotype-matched control rat mAbs (rat anti-mouse CD4 mAb GK1.5, rat anti-mouse CD8 mAb 2.43, or rat anti-human HLA mAb SFR3D5) were prepared as ascites fluids from hybridoma-inoculated, pristane-primed nu/nu mice (Bioproducts for Science, Indianapolis, IN). Abs were partially purified by precipitation with 50% ammonium sulfate, and dialyzed against PBS to a final concentration of 1 mg of IgG/ml as determined using anti-rat IgG immunodiffusion plates (ICN Pharmaceuticals, Costa Mesa, CA). Lymphocyte-depleting Abs contained in purified rat ascites were administered as 1 mg of Ab in a 1-ml volume i.p. on day 25 (3 days prechallenge), day 27 (1 day prechallenge), and day 30 (2 days postchallenge). The mAbs depleted >99% of the indicated lymphocyte population as determined using flow cytometry analysis, as described previously (29, 30, 31).
Timeline
The timing of administration of Abs and viruses and the days on
which tissue samples and body fluids were obtained for specific tests
are summarized in Fig 1
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of passively acquired RSV Abs on the immunogenicity and
efficacy of live attenuated RSV vaccine candidates was first studied
(Tables I and II). Two vaccine candidates with differing levels of
attenuation, cpts-248 and its more attenuated derivative
cpts-248/804, were selected because they might manifest
differing sensitivity to the immunosuppressive effects of RSV Abs. Two
concentrations of passive RSV Abs were studied to explore the range of
effects such Abs might have on the immunogenicity of the two vaccine
candidates for mice. Importantly, the level of RSV neutralizing Abs
achieved in the recipients of undiluted MIS on the day following
passive immunization was 1:320, a titer similar to that present on day
28 in animals infected with wt RSV (1:864). This indicates that
physiological levels of RSV Abs, i.e., titers that would be seen in
human infants within the first several months of life, were achieved in
the passively immunized animals.
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, group, G, columns 6 and
7), and for cpts-248 and cpts-248/804 (Table I,
groups C and F, columns 4 and 5). Passively acquired RSV Abs restricted
replication of the vaccine viruses in the lower, but not the upper
respiratory tract, and the level of restriction observed reflected the
amount of Ab transferred (Table I, groups A, B, D, and E, columns 4
and 5).
We next investigated the effect of passive Abs on the protective
efficacy of candidate vaccine viruses by challenging immunized animals
28 days postimmunization with wt RSV. A high level of protection
against challenge virus was noted in all groups of immunized animals,
even those that had received passive Abs (Table I, columns 6 and 7).
Resistance to virus replication in the lower respiratory tract was
observed even in those groups in which vaccine virus replication during
primary immunization was highly restricted in the lower respiratory
tract, with a lower degree of restriction observed in the NTs (Table I,
groups A, D, and E). Serologic analysis using RSV-F IgG ELISA or virus
neutralization assay demonstrated that passive Abs inhibited the
primary Ab response to immunization. In some cases, passive Abs
completely abrogated a detectable serum neutralizing Ab response, even
in groups that were resistant to challenge (Table II, groups A, B, D, and E). Analysis of
serologic responses to challenge indicated that secondary Ab response
patterns were usually noted (i.e., higher responses than that of the
control group (i.e, Table II, group G) on day 28 postimmunization
following primary wt virus infection) even in the absence of a prior
detectable primary Ab response (Table II, groups B and E). These data
indicate that priming of B lymphocytes occurred in the setting of
passive/active immunization, even when serum Ab responses were not
detected. However, in contrast to our passive/active immunization
studies in chimpanzees, the secondary response of passive/active
immunized mice was not enhanced compared with mice immunized without
passive Abs (21). The higher dose of passive Abs
significantly decreased both primary and secondary serum Ab responses,
but unexpectedly did not diminish protective efficacy (Table I, groups
A and D).
Mucosal Ab studies
We next studied (Table III) whether
the high level of resistance to RSV challenge manifested by the
passively/actively immunized mice in the absence of detectable serum
RSV neutralizing Abs could have been mediated by mucosal IgA Abs. The
studies were performed with the cpts-248 virus mice
passively administered NMS or MIS (corresponding to groups A and C of
Tables I and II) because this MIS-treated group exhibited complete
restriction of virus replication after challenge in the absence of a
detectable serum Ab response. Prior immunization with
cpts-248 in the absence of RSV Abs induced both local IgA
and IgG Abs in NW and BAL secretions (day 0 titers in middle rows
(NMS/cpts-248) of Table III). However, if passive Abs were
present at the time of immunization (MIS/cpts-248), these
local Ab responses were completely suppressed at the time of challenge.
We also measured local Ab responses on days 4 and 7 postchallenge to
determine whether an unusually rapid rise of local Abs occurred in the
MIS-treated group following challenge that might have inhibited
replication of the challenge virus. Such rises were not detected in the
MIS-treated group (Table III) or in control animals undergoing primary
infection (Table III). The absence of an IgA and IgG immune response in
mucosal secretions and serum in the MIS-treated/cpts-248
group (Table III) and the failure to recover the wt RSV challenge virus
(Table I, group A) indicated that the level of resistance induced by
passive/active immunization was high and still very active 28 days
after infection.
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) at the time of wt virus
challenge (day 28, also termed postchallenge day 0) and following
challenge (postchallenge days 4 and 7). Before infection there were few
if any RSV-specific AFC in any tissue, whereas previously immunized
animals had a large number of RSV-specific IgG AFC detected in
respiratory and lymphatic tissues, plus low to moderate levels of IgA
AFC. The numbers of AFC on day 0 of MIS-treated previously immunized
animals were similar to those of the control animals, indicating that
passive RSV Abs actively suppressed the RSV-specific AFC response to
immunization with the RSV vaccine candidate. In the nonimmunized group,
a significant increase of RSV-specific AFC was noted in the MLN on day
4, and in the lungs and spleen by day 7. Only few RSV-specific AFC were
noted in the tissues of passive/active immunized animals, and the
number of these cells was lower than that in the primary infection
group in which virus replication was not restricted. Thus, an
accelerated or augmented AFC response was not seen in the
passive/active immunization group, suggesting that Abs were not the
principal mediators of resistance to challenge in this group.
Interestingly, the AFC that were induced in the passive/active group
appeared in the MLN by day 4 then redistributed to the lungs 3 days
later.
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Because neither humoral Abs nor AFC appeared to be the mediators
of resistance to replication of RSV challenge virus in the
passive/active immunization group, we considered the possibility that T
cells are the principal mediators of resistance in this group. To test
this hypothesis, passive/active immunized mice were depleted of T cells
at the time of RSV challenge leaving humoral immune mechanisms, if
present, intact to restrict replication of RSV challenge virus. Mice
were pretreated with MIS or NMS and then were infected with the RSV
cpts-248 vaccine virus or a heterologous wt respiratory
virus (PIV3). At the time of wt RSV challenge 28 days after
immunization, CD4+, CD8+,
or CD4+ and CD8+ T cells
were depleted in vivo using depleting Abs (Table V). As expected, replication of RSV
challenge virus during primary infection (in the NMS-treated/PIV3
group) was not affected by the depletion of T cells.
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) would have developed
both RSV-specific Abs (shown in Tables II and III) as well as
RSV-specific T cells, each of which could mediate resistance to
replication of RSV challenge virus. Protection of the
NMS-treated/cpts-248 group in the lungs was most likely
mediated by the high levels of Abs induced by immunization (as shown in
Tables II-IV). Depletion of CD8+ and
CD4+ T cells resulted in a 20-fold increase in
replication in the NT of NMS-treated/cpts-248 animals, but
no increase in the lungs of these animals. This observation suggests
that RSV Abs completely protect the lungs, but local Abs plus local T
cell immunity protect the nasopharynx. Abs present in the upper
respiratory tract appear to provide a 10-fold reduction in challenge
virus replication, whereas the remainder of the reduction requires T
cells.
However, in passive/active immunized mice (the
MIS-treated/cpts-248 group in Table V), T cell depletion had
a major effect on protective efficacy in both the upper and lower
respiratory tract. CD4+ or
CD8+ T cell depletion alone had little effect on
protective efficacy in the NT or lungs. In contrast, simultaneous
depletion of both CD4+ and
CD8+ lymphocytes completely abrogated protection
in the NT and allowed a 100-fold higher level of replication in the
lungs. It appears that CD4+ and
CD8+ T cells each make independent contributions
to this immunity. These data suggest that CD4+
and CD8+ T cells are the major mediators of
resistance to challenge in the NT of passive/active immunized mice,
whereas T cells and possibly a low level of Abs or other humoral
factors protect the lower respiratory tract in passive/active
immunization. The mediator of resistance accounting for the residual
90-fold restriction of replication in lungs in
CD4+/CD8+ T cell depleted
passive/active immunized mice was not clear; however, we suggest that
the low level of AFC (Table IV) detected in lung may contribute to this
effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current studies in mice show that live RSV vaccine candidates can readily induce immunity even when immunization occurs in the presence of physiologic levels of passively acquired RSV neutralizing Abs. Although passive Ab suppresses the generation of serum and mucosal RSV-specific Abs and the RSV-specific AFC response, a high level of immunity was seen in the mice. As indicated above, passively acquired serum IgG Abs are known to suppress the active IgG serum Ab response to immunization, but much less is known about the effect of such passive Abs on the mucosal immune response to respiratory tract infection (43). In the present study, the passively acquired RSV Abs suppressed not only systemic Ab responses induced by immunization with live attenuated RSV vaccines, but also suppressed the mucosal IgA, IgM, and IgG RSV-specific Ab responses as well as the regional and systemic Ab-secreting cell responses. These observations identified a broad suppression of the humoral arm of the immune response and suggested that T cells contributed to the immunity observed in the passively/actively immunized mice. Indeed, CD4+ and CD8+ T cells each were found to make significant independent contributions to resistance to replication of RSV in those mice whose primary Ab response had been suppressed by passive Abs. In such mice, humoral immunity also contributed to the restriction of replication of RSV challenge virus in the lower respiratory tract, whereas in the upper respiratory tract, the CD4+ and CD8+ T cells were identified as the sole mediators of the observed resistance. The ability of CD4+ and CD8+ T cells induced by immunization to restrict replication of RSV has been described previously (29, 44). At the other end of the spectrum, mice immunized with the live RSV vaccine candidate in the absence of passively acquired RSV neutralizing Abs were also highly resistant to replication of RSV challenge virus, but this resistance was only slightly diminished by depletion of CD4+ and CD8+ T cells at the time of challenge, indicating that resistance was mediated by Abs. Because a high level of resistance to replication of RSV challenge virus was seen in passively/actively immunized mice over a 10-fold range of passively acquired RSV Ab concentrations, it appears that the relative contribution of the humoral and cellular arms of the immune response to overall immunity varies depending on the extent of suppression of the primary Ab response by the passively acquired RSV neutralizing Abs.
It was somewhat surprising to identify CD4+ and CD8+ T cells as major mediators of resistance to reinfection with RSV. T cells are important for resolution of acute RSV infection (45). However, they have been identified infrequently as contributors to protection against reinfection with RSV, and the resistance mediated by RSV-specific CD8+ T cells appears short-lived (2). The T cell-mediated mechanism of resistance identified in this paper may apply to other virus infections of infants who require immunization in the presence of maternal Abs, such as measles virus and influenza virus (41, 46, 47). Such studies with other viruses taken together with our data suggest that immunization with live respiratory viruses mediate protection by a novel T cell mechanism requiring the cooperation of CD4+ and CD8+ T cells when the usual mechanism of protection, i.e., neutralizing Abs, is suppressed. Because the duration of resistance against a respiratory virus mediated by effector T cells is generally of short duration (2, 48, 49), i.e., measured in weeks or months rather than years, it is likely that effective durable immunity in the neonatal period will require readministration of vaccine until a sustained serum and mucosal Ab response is induced.
One needs to exercise caution in extrapolating the findings from the present study in mice to the analogous situation in humans. In humans, administration of a live attenuated RSV vaccine to 1-mo-old infants possessing maternally acquired RSV Abs induced a high level of resistance to replication of a second dose of vaccine virus given 1 mo later (50). Similar to the data presented here, very few of those infants exhibited a serum RSV-specific IgG or neutralizing Ab response following immunization in the presence of RSV-specific maternal Abs. In contrast to passively/actively immunized mice, which failed to develop a systemic or mucosal Ab response to immunization with live RSV vaccine, most human infants possessing levels of maternally acquired RSV Abs similar to those observed in mice in the present study, developed a moderate level of serum and NW RSV IgA Abs. In these infants, the resistance to replication of the second dose of RSV vaccine virus correlated with the presence of an Ab response to the RSV G glycoprotein. Therefore, in humans the humoral immune response may be of importance in the very young infant. It is unclear why humans and mice differ in their responses to passive/active RSV immunization in this regard. One factor may be the observation that RSV infection of mice is only semipermissive, making it easier for a weak or a minor immune response to RSV to restrict its replication effectively upon reinfection.
The mechanism of Ab-mediated immune suppression in human neonates has not been fully defined. Passively acquired Abs may simply mediate blocking or accelerated clearance of immunizing Ag by determinant-specific binding (51). Nevertheless, data from experimental systems suggest that Ag-Ab immune complexes formed in vivo can have potent immunoregulatory, including suppressive, effects, when such diverse Ags as pneumococcal polysaccharides (52), Listeria monocytogenes organisms (53), and model Ags like diphtheria and tetanus toxins, sheep red blood cells, bacteriophages, and mammalian viruses are studied (54). Possible mechanisms of Ab-mediated immunoregulation include: 1) Ab-Ag immune complex inhibition of B cell receptor signaling by coligation of the receptor for the Fc region of IgG (FcRIIB1) and the B cell receptor (55), and 2) inhibition of cytokine-mediated induction of major histocompatibility complex expression in APC (56).
When primary Ab responses are suppressed by passive Abs, priming of the B cell repertoire for secondary responses of greater magnitude and quality (i.e., a memory response with enhanced neutralizing activity) may still occur. This observation was made previously in RSV passive/active immunization studies in chimpanzees and bovines (21, 57, 58). Inhibition of primary Ab response with sparing of the secondary response has also been noted in experimental systems using Ag-Ab complexes, for example, diphtheria toxin-antitoxin precipitates (54). Whether this phenomenon also occurs in humans has not been adequately evaluated. The unusual phenomenon of significant enhancement of secondary Ab responses by prior passive/active immunization that we previously noted in chimpanzees (21) was not seen in the present study in the mouse. In the primate studies, the primary serum Ab response to live RSV i.n. immunization as measured by ELISA binding or neutralizing Abs was decreased at least 2-fold. Surprisingly, the secondary Ab response to challenge 1 mo following passive/active immunization was greatly enhanced both in quantity (absolute titer) and quality (ratio of neutralizing to binding Ab reciprocal titer). The mechanism underlying this heightened secondary Ab response in primates remains unexplained. There is no published data to suggest that RSV infection of human infants in the presence of passively acquired RSV Abs primes for enhanced secondary responses. In this respect, the mouse studies reported here may better reflect the dynamics of immune response in humans infected in the presence of passively acquired Abs.
These studies in mice demonstrate that passively acquired RSV Abs suppress both systemic and local Ab responses to primary infection with live attenuated RSV vaccine candidates. However, this Ab-mediated immune suppression does not abrogate the development of resistance to wt virus challenge 1 mo later, which was shown to be mediated by both CD4+ and CD8+ T cells. Despite inhibition of the primary humoral response, passive Abs generally did not affect priming for a secondary Ab response following challenge. Successful local and systemic immunity has been induced in young infants with maternal Abs who were immunized with other live vaccines such as live poliovirus or live rotavirus vaccines, but a multidose schedule of administration is required to achieve optimal immunization. Our studies suggest that an effective immunization strategy for RSV in young human infants who possess maternal RSV Abs likely also will require multiple doses of vaccine early in life.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: RSV, respiratory syncytial virus; NMS, nonimmune mouse serum; MIS, mouse immune serum; NT, nasal turbinate; wt, wild type; AFC, Ab-forming cell; i.n., intranasal; BAL, bronchoalveolar lavage; F, fusion protein; G, attachment protein; NW, nasal wash; CLN, cervical lymph node; MLN, mediastinal lymph nodes; NALT, nasal associated lymphoid tissue; PIV3, parainfluenza virus type 3; cpts, cold-passaged temperature-sensitive; GMT, geometric mean titer.
Received for publication January 23, 2001. Accepted for publication August 7, 2001.
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 P. G. Thomas, S. A. Brown, W. Yue, J. So, R. J. Webby, and P. C. Doherty An unexpected antibody response to an engineered influenza virus modifies CD8+ T cell responses PNAS, February 21, 2006; 103(8): 2764 - 2769. [Abstract] [Full Text] [PDF]
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 P. L. Collins and B. R. Murphy New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics Proceedings of the ATS, August 1, 2005; 2(2): 166 - 173. [Abstract] [Full Text] [PDF]
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 S. Mrusek, S. Vallbracht, and S. Ehl The impact of splenectomy on antiviral T cell memory in mice Int. Immunol., January 1, 2005; 17(1): 27 - 33. [Abstract] [Full Text] [PDF]
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