Passively Acquired Antibodies Suppress Humoral But Not Cell-Mediated Immunity in Mice Immunized with Live Attenuated Respiratory Syncytial Virus Vaccines This information is current as of September 25, 2021. James E. Crowe, Jr., Cai-Yen Firestone and Brian R. Murphy J Immunol 2001; 167:3910-3918; ; doi: 10.4049/jimmunol.167.7.3910 http://www.jimmunol.org/content/167/7/3910 Downloaded from

References This article cites 57 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/167/7/3910.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 25, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Passively Acquired Antibodies Suppress Humoral But Not Cell-Mediated Immunity in Mice Immunized with Live Attenuated Respiratory Syncytial Virus Vaccines

James E. Crowe, Jr.,1* Cai-Yen Firestone,† and Brian R. Murphy†

A respiratory syncytial virus (RSV) vaccine will need to be administered by 1 mo of age to protect young infants; therefore, it will need to be effective in the presence of maternally acquired RSV Abs. In the present study, the immunogenicity and efficacy of two live attenuated RSV vaccine candidates of different level of attenuation were evaluated in mice passively immunized with varying quantities of RSV Abs. The replication of the RSV vaccines was suppressed in the lower, but not the upper, respiratory tract of the passively immunized mice. Immunization with either vaccine candidate was highly efficacious against challenge with wild-type

RSV in both passively immunized and control mice. Nonetheless, a high level of immunity was seen even in passively/actively Downloaded from immunized animals that failed to develop a humoral immune response, suggesting that T cells mediated the immunity. Depletion of CD4؉ and CD8؉ T cells in passively/actively immunized and control animals at the time of challenge with wild-type RSV demonstrated that CD4؉ and CD8؉ T cells made significant independent contributions to the restriction of replication of RSV challenge virus in both the upper and lower respiratory tracts. Although passively acquired serum RSV Abs suppressed the primary systemic and mucosal Ab responses of IgM, IgG, and IgA isotypes, B lymphocytes were nevertheless primed for robust ؉ ؉ secondary Ab responses. Thus, immunity mediated by CD4 and CD8 T cells and Abs can be readily induced in mice by live http://www.jimmunol.org/ RSV vaccine candidates in the presence of physiologic levels of RSV neutralizing Abs. The Journal of Immunology, 2001, 167: 3910–3918.

espiratory syncytial virus (RSV)2 is the major cause of 6). The relative sparing of infants from virus-associated lower re- serious viral lower respiratory tract illness in infants and spiratory tract disease in the first weeks or months of life correlates R young children throughout the world (1). In the devel- with the level of passively acquired maternal Abs to RSV (7), oped world, RSV causes serious lower respiratory tract disease parainfluenza virus type 3 (PIV3) (8), or influenza viruses (9). necessitating hospitalization, often requiring intensive medical Administration of pooled human IgG containing a high titer of care, in 0.5–1% of the population Ͻ1 year of age (1). The peak RSV neutralizing Abs or of a humanized murine RSV-specific by guest on September 25, 2021 incidence of severe disease occurs at a very young age when most mAb prevents severe RSV disease in high-risk infants and children infants still possess maternally acquired RSV Abs. The immuno- (10, 11). Passive transfer of RSV polyclonal immune sera or neu- logic mechanisms that mediate protection against RSV infection tralizing RSV mAbs protects the lower, and to a lesser extent, the and disease are under investigation. CTLs contribute to the reso- upper respiratory tract of rodents against replication of RSV chal- lution of infection by respiratory viruses; however, their rapid dis- lenge virus (12–15). The F (fusion) and G (attachment) glycopro- appearance following clearance of virus, and the delay in their teins are the major protective Ags of RSV, and only these RSV activation following virus reinfection, suggest that they do not play Ags induce neutralizing Abs (2). A major goal of active RSV a major role in the prevention of reinfection by respiratory viruses immunization is to induce high levels of serum and mucosal neu- (2–4). tralizing Abs that are able to protect the upper (16, 17) and lower Clinical and laboratory studies provide strong evidence that respiratory tract against a high level of replication of wild-type Abs, including mucosal and serum Abs, play a dominant role in (wt) RSV. protection against infection or reinfection by respiratory viruses (5, Unfortunately, infants in the first 6 mo of life mount a poor primary Ab response to respiratory virus infection or immuniza- *Department of Pediatrics, Vanderbilt University, Nashville, TN 37232; and †Respi- tion (5, 6). The overall level of serum and mucosal Ab response to ratory Viruses Section, Laboratory of Infectious Diseases, National Institute of Al- the F and G glycoproteins of young infants to primary RSV in- lergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 fection is ϳ15–25% that of older infants and children (18, 19). Received for publication January 23, 2001. Accepted for publication August 7, 2001. Two principal factors contribute to this diminished response, The costs of publication of this article were defrayed in part by the payment of page namely, maternal Ab-mediated immune suppression and immuno- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. logical immaturity (18). A high level of maternally acquired RSV Abs decreased the serum Ab response to the G glycoprotein, and 1 Address correspondence and reprint requests to Dr. James E. Crowe, Jr., Division of Pediatric Infectious Diseases, Department of Pediatrics, Vanderbilt University Med- young age diminished the response to the F glycoprotein in infants ical Center, D-7235 MCN, 1161 21st Avenue S., Nashville, TN 37232-2581. E-mail with RSV infection (18). In experimental animals, passive transfer address: [email protected] of RSV Abs decreased the frequency and magnitude of the Ab 2 Abbreviations used in this paper: RSV, respiratory syncytial virus; NMS, nonim- response to F and G proteins expressed by vaccinia virus recom- mune mouse serum; MIS, mouse immune serum; NT, nasal turbinate; wt, wild type; AFC, Ab-forming cell; i.n., intranasal; BAL, bronchoalveolar lavage; F, fusion pro- binants and decreased the neutralizing activity of the Ab that was tein; G, attachment protein; NW, nasal wash; CLN, cervical lymph node; MLN, induced (20). In seronegative chimpanzees, passive RSV Abs sup- mediastinal lymph nodes; NALT, nasal associated lymphoid tissue; PIV3, parainflu- enza virus type 3; cpts, cold-passaged temperature-sensitive; GMT, geometric mean pressed the primary Ab response to infection with live attenuated titer. RSV virus vaccine candidates but, unexpectedly, did not inhibit

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3911 the development of resistance to wt virus challenge or the second- cells (28); plaques were visualized using the RSV-specific immunohisto- ary Ab response to infection with RSV challenge virus (21). The logical staining procedure (26). Neutralizing Ab titers were expressed as mechanisms underlying the protective efficacy of live attenuated the reciprocal of the highest Ab (or serum) dilutions that yielded a 60% reduction in PFU as compared with the control wells. vaccines in chimpanzees immunized in the presence of passively ELISPOT assay. Murine Ab-forming cells (AFC) secreting RSV-F-spe- acquired RSV Abs have not been defined. The diminished Ab re- cific Abs of the IgM, IgA, or IgG isotypes were quantitated in an ELISPOT sponse to immunization suggested that other immune mediators, assay. Flat-bottom Immulon I immunoassay plates (Nunc, Naperville, IL) such as T cells, might contribute to the observed resistance to were coated overnight with 200 ng/well immunoaffinity-purified RSV F replication of RSV challenge virus. glycoprotein in carbonate buffer, pH 9. Plates were washed with buffer, and In the present study, the immunogenicity and efficacy of two serial 2-fold dilutions of harvested cell populations in RPMI 1640 medium live attenuated RSV vaccine candidates differing in their level of 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 attenuation were evaluated in mice passively immunized with blocked with PBS/1% BSA. RSV F protein-specific Abs secreted by the varying quantities of RSV Abs. The live RSV vaccine candidates cells were detected as spots using peroxidase-labeled isotype-specific goat induced a high level of immunity in mice, as in chimpanzees (21), anti-mouse Abs (Southern Biotechnology Associates, Birmingham, AL) at when immunization was performed in the presence of physiolog- a 1/1000 dilution in PBS/1% BSA, incubated overnight, then washed three ical levels of passively transferred RSV neutralizing Abs. Immu- times with PBS/0.05% Tween 20 and deionized water, and overlaid with 50 ␮l/well substrate (a 1:1 mixture of 2% low-melting-point agarose and nity exhibited by these mice was shown in the present study to be ϩ ϩ ϩ NBT/BCIP substrate solution; Sigma, St. Louis, MO). Spots were enumer- mediated by CD4 and CD8 T cells as well as by Abs. CD4 ated under a dissecting microscope, and expressed as number of AFC per and CD8ϩ T cells made a greater contribution in those mice with 106 total cells. a high level of Ab-mediated suppression of the humoral immune Preparation of RSV immune and nonimmune serum. Twenty-four- Downloaded from response, whereas humoral immunity played the major role in week-old BALB/c mice were infected by the intranasal (i.n.) route with 6.3 those mice with a strong humoral immune response. 10 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 Materials and Methods 56°C for 30 min. The serum pool had a neutralizing Ab titer of 1:20,000. Cell culture This serum pool was diluted 1/5 in sterile PBS to generate the working

suspension for passive immunization designated “mouse immune serum” http://www.jimmunol.org/ HEp-2 cells, obtained from the American Type Culture Collection (Man- (MIS). Control BALB/c mice of an identical age that were not infected assas, VA) at passage 364, were maintained as previously described (22) with RSV were bled concomitantly to generate a nonimmune serum pool and were not used beyond passage 400. HEp-2 cell monolayer cultures Ͻ grown on 24-well tissue culture plates (Costar) were used for all virus that had a RSV neutralizing Ab titer of 1:20. This serum pool was like- assays as previously described (22). wise heat-inactivated and diluted 1/5 with PBS to generate the working suspension designated “nonimmune mouse serum” (NMS). Viruses 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 The wt RSV strain A2 and the isolation and characterization of the cold- passaged temperature-sensitive (cpts)-248 and cpts-248/804 mutants were with PBS alone on the day before primary i.n. immunization with virus. described previously (23–25). Briefly, the wt RSV strain A2 was multiply Data from groups treated with MIS 1/10 and MIS 1/100 were similar, and passaged at low temperature to yield a host-range mutant, designated cp- data from the groups treated with NMS or PBS were similar; therefore, by guest on September 25, 2021 RSV, that was subsequently chemically mutagenized to yield a tempera- only the data from MIS 1/10 and NMS are shown. ture-sensitive (ts) mutant designated cpts-248. The shut-off temperature of Active immunizations. Mice were immunized by the i.n. route on day 0 cpts-248 (lowest temperature at which a 100-fold reduction in plaque titer with 106.5 PFU of virus in a 0.1-ml inoculum. The viruses used were RSV vs titer at permissive temperature occurs) was 38°C. The cpts-248 mutant cpts-248, cpts-248/804, wt RSV strain A2, or a heterologous control virus was then mutagenized to yield a ts mutant with a lower shutoff temperature, (wt PIV3 strain JS) as indicated in Tables I-V. On day 4 following inoc- cpts-248/804 (37°C shutoff). Each of the viruses was biologically cloned ulation, a subset of mice from each treatment group was sacrificed by CO2 by three plaque-to-plaque passages in Vero cells and subsequently ampli- inhalation, and NT and lung tissues were harvested for quantitation of virus fied in Vero cell monolayer culture. These mutants differed in their repli- and characterization of recovered virus as described (24). Twenty-eight cative capacity in rodents or chimpanzees, with cpts-248 replicating to a days following the initial virus infection, mice were challenged i.n. under high level, whereas cpts-248/804 replicated to an intermediate level (24, anesthesia with 106.3 PFU of wt RSV strain A2 in a 0.1-ml inoculum. On 25). Each virus was concentrated by ultracentrifugation as previously de- day 4 following challenge, a subset of three mice from each treatment scribed (25) to achieve the high virus input titer needed in experiments group was sacrificed, and NT and lung tissues were obtained for virus performed in mice. A similarly prepared wt PIV3 (strain JS) suspension titration. Groups of six mice were bled postinfusion (day 0), postimmuni- was used as a heterologous virus control in some experiments. zation (day 28), and postchallenge (day 56) to obtain serum for assay of Virus titrations neutralizing or ELISA Ab titer. Regional Ab and AFC samples. Subsets of animals from each treatment

Virus present in tissue culture harvests or in the lung or nasal turbinate group were sacrificed by CO2 inhalation, and serum, bronchoalveolar la- (NT) homogenates of mice was quantitated by plaque assay on HEp-2 cell vage (BAL) fluid, and nasal wash (NW) fluid were collected on day 28 monolayer cultures maintained under a semisolid methylcellulose overlay (before challenge), or on days 32, 35, or 56 (days 4, 7, or 28 after wt virus at 37°C as described, except that plaque assays of the ts mutants were challenge) to determine the level of RSV-specific Abs present. Blood was performed at the permissive temperature of 32°C (22). Staining monolay- obtained by heart puncture. BAL fluids were obtained by ligation of the ers with RSV-specific murine mAbs using an immunoperoxidase proce- trachea with suture, insertion of a 23-gauge blunt needle into the distal dure (26) facilitated detection of plaques. trachea, followed by three in-and-out flushes of the airways with 3 ml of Immunological studies 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 Animals. Respiratory-pathogen-free BALB/c mice were obtained from NW were concentrated 10-fold using 50 kDa molecular mass cutoff spin Charles River Breeding Laboratories (Wilmington, MA) and were studied concentrators (Millipore, Bedford, MA). at 24–32 wk of age. Mice within each experiment were age-matched. An- Lymphocyte populations were harvested from lymphoid tissues on days imals were maintained in microisolator cages throughout the studies. All 28, 32, or 35 for quantitation of RSV-specific AFC using the ELISPOT experiments involving animals were reviewed and approved by the Na- technique. Cells were pooled from groups of five (day 28) or three animals tional Institutes of Health Animal Care and Use Committee. (days 32 and 35). Total cells were obtained from cervical lymph nodes Serology. Serum IgG Abs binding to RSV F surface glycoprotein were (CLN), mediastinal lymph nodes (MLN), or spleen by passage of the entire quantitated in an ELISA using F glycoprotein that had been immunoaffin- organ through a sterile wire mesh screen into RPMI 1640 with 5% FCS, 50 ity-purified from RSV subgroup A (Long strain)-infected cell lysates as ␮g/ml gentamicin, and 2.5 ␮g/ml amphotericin (cRPMI). Nasal associated described (19, 27). The neutralizing Ab titer of serum samples was deter- lymphoid tissue (NALT) was obtained by dividing the soft and hard palates mined by a complement-enhanced 60% plaque reduction assay in HEp-2 through the midline, then gently scraping all visible soft tissue into cRPMI, 3912 RSV Abs SUPPRESS HUMORAL BUT NOT T CELL-MEDIATED IMMUNITY followed by passage through a wire mesh screen. Mononuclear cell frac- studied (Tables I and II). Two vaccine candidates with differing tions were obtained from the lungs as follows. The pulmonary vasculature levels of attenuation, cpts-248 and its more attenuated derivative was cleared of blood by in situ flushing with 3–5 ml of cold sterile PBS cpts-248/804, were selected because they might manifest differing administered via 23-gauge needle inserted into the right ventricle. The complete lungs were removed and immersed in cRPMI containing DNase sensitivity to the immunosuppressive effects of RSV Abs. Two I (Sigma) and collagenase type III (Worthington Biochemical, Lakewood, concentrations of passive RSV Abs were studied to explore the NJ), minced into fine pieces with surgical scissors, then shaken gently at range of effects such Abs might have on the immunogenicity of the 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 (Lym- two vaccine candidates for mice. Importantly, the level of RSV pholyte M; CEDARLANE Laboratories, Hornby, Ontario, Canada), and neutralizing Abs achieved in the recipients of undiluted MIS on the centrifuged at 400 ϫ g for 20 min. The mononuclear cell fraction obtained day following passive immunization was 1:320, a titer similar to from the gradient interface was resuspended and washed three times in that present on day 28 in animals infected with wt RSV (1:864). cRPMI. The cell count of the final suspensions of NALT, CLN, MLN, lung cells, or spleen cells was determined using a microscope and hemacytom- This indicates that physiological levels of RSV Abs, i.e., titers that eter. The resulting cell suspensions were used in the ELISPOT assay. would be seen in human infants within the first several months of life, were achieved in the passively immunized animals. T cell depletion studies In mice that had not received RSV Abs, a gradient of replication Lymphocyte-depleting or isotype-matched control rat mAbs (rat anti- (from high to low) in the upper and lower respiratory tract was mouse CD4 mAb GK1.5, rat anti-mouse CD8 mAb 2.43, or rat anti-human observed for wt RSV (Table I, group, G, columns 6 and 7), and for HLA mAb SFR3D5) were prepared as ascites fluids from hybridoma-in- oculated, pristane-primed nu/nu mice (Bioproducts for Science, Indianap- cpts-248 and cpts-248/804 (Table I, groups C and F, columns 4

olis, IN). Abs were partially purified by precipitation with 50% ammonium and 5). Passively acquired RSV Abs restricted replication of the Downloaded from sulfate, and dialyzed against PBS to a final concentration of 1 mg of IgG/ml vaccine viruses in the lower, but not the upper respiratory tract, as determined using anti-rat IgG immunodiffusion plates (ICN Pharmaceu- and the level of restriction observed reflected the amount of Ab ticals, 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 transferred (Table I, groups A, B, D, and E, columns 4 and 5). 25 (3 days prechallenge), day 27 (1 day prechallenge), and day 30 (2 days We next investigated the effect of passive Abs on the protective postchallenge). The mAbs depleted Ͼ99% of the indicated lymphocyte efficacy of candidate vaccine viruses by challenging immunized population as determined using flow cytometry analysis, as described

animals 28 days postimmunization with wt RSV. A high level of http://www.jimmunol.org/ previously (29–31). protection against challenge virus was noted in all groups of im- Timeline munized animals, even those that had received passive Abs (Table The timing of administration of Abs and viruses and the days on which I, columns 6 and 7). Resistance to virus replication in the lower tissue samples and body fluids were obtained for specific tests are sum- respiratory tract was observed even in those groups in which vac- marized in Fig 1. cine virus replication during primary immunization was highly re- stricted in the lower respiratory tract, with a lower degree of re- Results striction observed in the NTs (Table I, groups A, D, and E). Effect of passively acquired serum on replication of RSV Serologic analysis using RSV-F IgG ELISA or virus neutralization The effect of passively acquired RSV Abs on the immunogenicity assay demonstrated that passive Abs inhibited the primary Ab re- by guest on September 25, 2021 and efficacy of live attenuated RSV vaccine candidates was first sponse to immunization. In some cases, passive Abs completely

FIGURE 1. Timeline indicating elements of the experimental plan. Materials administered are shown above the timeline, tests performed on indicated tissue or body fluid sample are shown below the timeline. Passive Abs and virus inoculations were administered and virus titrations performed in every experiment. Other tests were performed in separate experiments as indicated in the tables. Neut. Ab., Neutralizing Abs; Lg, lung; S, serum; Sp, spleen. The Journal of Immunology 3913

Table I. Passively acquired RSV Abs suppress pulmonary virus replication following infection with a live attenuated RSV vaccine candidate, but do not abrogate resistance to subsequent wt virus challenge

Virus Titer (mean log10 PFU/g of three animals) on Day 4 in Indicated Tissue After Inoculation with Immunizing or Challenge Virus

Serum Immunizing virus Challenge virus Immunizing Infused Virusa Group Day Ϫ1 NT Lungs NT Lungs

cpts-248 A MISb 3.4 2.2 Ͻ2.0 Ͻ1.7 B MIS 1/10c 3.4 4.2 2.2 1.9 C NMSd 3.5 5.2 Ͻ2.0 Ͻ1.7 cpts-248/804 D MIS Ͻ2.0 Ͻ1.7 2.2 1.9 E MIS 1/10 2.5 2.5 2.5 1.8 F NMS 2.7 3.1 2.1 Ͻ1.7 wt RSV A2 G None nde nd 4.9 6.2

a The live attenuated or wt RSV immunizing virus was given i.n. at a dose of 106.5 PFU/mouse on day 0; the wt challenge virus was given i.n. on day 28. b BALB/c mouse RSV-immune serum. c MIS diluted 1/10 in PBS.

d BALB/c mouse RSV-nonimmune serum. Downloaded from e nd, Not done. abrogated a detectable serum neutralizing Ab response, even in NMS or MIS (corresponding to groups A and C of Tables I and II) groups that were resistant to challenge (Table II, groups A, B, D, because this MIS-treated group exhibited complete restriction of and E). Analysis of serologic responses to challenge indicated that virus replication after challenge in the absence of a detectable se- secondary Ab response patterns were usually noted (i.e., higher rum Ab response. Prior immunization with cpts-248 in the absence http://www.jimmunol.org/ responses than that of the control group (i.e, Table II, group G) on of RSV Abs induced both local IgA and IgG Abs in NW and BAL day 28 postimmunization following primary wt virus infection) secretions (day 0 titers in middle rows (NMS/cpts-248) of Table even in the absence of a prior detectable primary Ab response III). However, if passive Abs were present at the time of immu- (Table II, groups B and E). These data indicate that priming of B nization (MIS/cpts-248), these local Ab responses were completely lymphocytes occurred in the setting of passive/active immuniza- suppressed at the time of challenge. We also measured local Ab tion, even when serum Ab responses were not detected. However, responses on days 4 and 7 postchallenge to determine whether an in contrast to our passive/active immunization studies in chimpan- unusually rapid rise of local Abs occurred in the MIS-treated group zees, the secondary response of passive/active immunized mice following challenge that might have inhibited replication of the by guest on September 25, 2021 was not enhanced compared with mice immunized without passive challenge virus. Such rises were not detected in the MIS-treated Abs (21). The higher dose of passive Abs significantly decreased both primary and secondary serum Ab responses, but unexpectedly group (Table III) or in control animals undergoing primary infec- did not diminish protective efficacy (Table I, groups A and D). tion (Table III). The absence of an IgA and IgG immune response in mucosal secretions and serum in the MIS-treated/cpts-248 group Mucosal Ab studies (Table III) and the failure to recover the wt RSV challenge virus We next studied (Table III) whether the high level of resistance to (Table I, group A) indicated that the level of resistance induced by RSV challenge manifested by the passively/actively immunized passive/active immunization was high and still very active 28 days mice in the absence of detectable serum RSV neutralizing Abs after infection. could have been mediated by mucosal IgA Abs. The studies were These findings suggested that both the mucosal immune re- performed with the cpts-248 virus mice passively administered sponse and the serum Ab response were suppressed in the passive/

Table II. Passively-acquired RSV Abs suppress primary serum Ab response to infection with a live attenuated RSV vaccine candidate and secondary serum Ab response to wt virus challenge, with the pattern of suppression varying with dose of passive Abs

Serum IgG RSV F Protein ELISA Titer (reciprocal mean Serum Neutralizing Ab Titer Against wt RSV A2 Ϯ log2 of six animals) (reciprocal GMT SEM of six animals) Serum Immunizing Infused Postinfusion Postimmunization Postchallenge Postinfusion Postimmunization Postchallenge Virusa Group Day Ϫ1 (day Ϫ1) (day 28) (day 56) (day Ϫ1) (day 28) (day 56)

cpts-248 A MISb 13.8 12.8 10.8 320 Ϯ 51 Ͻ40 Ͻ40 B MIS 1/10c 9.8 10.8 15.8 Ͻ40 Ͻ40 2239 Ϯ 1078 C NMSd 7.3e 13.3 16.8 Ͻ40 655 Ϯ 291 4719 Ϯ 1299 cpts-248/804 D MIS 12.3 10.3 11.3 319 Ϯ 75 Ͻ40 94.5 Ϯ 42 E MIS 1/10 10.3 8.3 13.8 Ͻ40 Ͻ40 1596 Ϯ 1476 F NMS 8.3e 12.8 16.8 Ͻ40 52.5 Ϯ 10 2684 Ϯ 798 wt RSV A2 G None ndf nd nd Ͻ40 864 Ϯ 279 3562 Ϯ 1785

a The live attenuated or wt RSV immunizing virus was given i.n. at a dose of 106.5 PFU/mouse on day 0, and the wt challenge was administered i.n. on day 28. b BALB/c mouse RSV-immune serum. c MIS diluted 1/10 in PBS. d BALB/c mouse RSV-nonimmune serum. e Indicates background levels of Ab binding to the RSV F glycoprotein seen in serum from unimmunized, naive animals. f nd, Not done. 3914 RSV Abs SUPPRESS HUMORAL BUT NOT T CELL-MEDIATED IMMUNITY

Table III. Both local secretory and serum Abs induced by infection with RSV cpts-248 are suppressed when primary infection occurs in the presence of passively acquired RSV Abs

RSV F Protein ELISA Titer on Indicated Day Following wt RSV A2 Challenge Serum (reciprocal GMT Ϯ SEMb) Infused on Immunizing Ab Day Ϫ1 Virusa Sample Isotype Day 0 Day 4 Day 7 Day 28

MIS cpts-248 NW IgA 10 Ϯ 0 Ͻ10 10 Ϯ 0nd IgG 10 Ϯ 0c 20 Ϯ 010Ϯ 0nd BAL IgA 16 Ϯ 6 Ͻ10 10 Ϯ 0nd IgG 10 Ϯ 010Ϯ 020Ϯ 10 nd Serum IgA 10 Ϯ 0nd10Ϯ 010Ϯ 0 IgG 2,560 Ϯ 1,920 nd 1,080 Ϯ 760 1,920 Ϯ 405 NMS cpts-248 NW IgA 16 Ϯ 610Ϯ 010Ϯ 0nd IgG 160 Ϯ 120 230 Ϯ 205 280 Ϯ 183 nd BAL IgA 208 Ϯ 11 160 Ϯ 0 440 Ϯ 200 nd IgG 928 Ϯ 418 1,280 Ϯ 640 3,840 Ϯ 3,200 nd Serum IgA 16 Ϯ 6nd10Ϯ 020Ϯ 10 IgG 108,544 Ϯ 34,208 nd 20,480 Ϯ 10,240 122,880 Ϯ 25,905 NMS None NW IgA 10 Ϯ 0 Ͻ10 10 Ϯ 0nd IgG 10 Ϯ 010Ϯ 010Ϯ 0nd BAL IgA 10 Ϯ 0 Ͻ10 10 Ϯ 0nd Downloaded from IgG 10 Ϯ 020Ϯ 10 Ͻ10 nd Serum IgA Ͻ10 nd 10 Ϯ 010Ϯ 10 IgG 904 Ϯ 430d nd 1,280 Ϯ 640 30,720 Ϯ 6,476

a Immunizing virus (106.5 PFU/mouse i.n.) was given on day 0 and challenge virus (106.3 PFU/mouse i.n.) was given 28 days later. b Groups of six animals. c The lower limit of detection was 10. For the purposes of calculation of reciprocal mean titers in groups for which at least one animal exhibited a reciprocal titer Ն10, the samples without reactivity were assigned a value of 10. http://www.jimmunol.org/ d This level of reactivity of IgG Ab in serum was considered background in this sensitive ELISA. active immunized group. We sought additional evidence to con- phatic tissues, plus low to moderate levels of IgA AFC. The num- firm the absence of mucosal Ab responses in the passive/active bers of AFC on day 0 of MIS-treated previously immunized ani- immunized groups by examining the level of RSV-specific IgM, mals were similar to those of the control animals, indicating that IgG, or IgA AFC in NALT, CLN, MLN, lungs, or spleen (Table passive RSV Abs actively suppressed the RSV-specific AFC re- IV) at the time of wt virus challenge (day 28, also termed post- sponse to immunization with the RSV vaccine candidate. In the challenge day 0) and following challenge (postchallenge days 4 nonimmunized group, a significant increase of RSV-specific AFC by guest on September 25, 2021 and 7). Before infection there were few if any RSV-specific AFC was noted in the MLN on day 4, and in the lungs and spleen by day in any tissue, whereas previously immunized animals had a large 7. Only few RSV-specific AFC were noted in the tissues of pas- number of RSV-specific IgG AFC detected in respiratory and lym- sive/active immunized animals, and the number of these cells was

Table IV. The number of IgM, IgG, and IgA AFCs is suppressed throughout the respiratory tract following primary RSV infection that occurs in the presence of passively acquired RSV Abs

No. of AFCs Secreting Abs Against RSV F Protein per 106 Cells on Virus Titer in Indicated Tissue Indicated Day of Challenge with wt RSV Serum 4 Days Following wt RSV a Infused Challenge (log10 PFU/g) Cells Harvested Day 0 Day 4 Day 7 on Day Immunizing from Indicated Ϫ1 Virus NT LungsSiteb IgM IgG IgA IgM IgG IgA IgM IgG IgA

MIS cpts-248 2.4 (4/6)c Ͻ1.7 (0/6)c NALT Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ11Ͻ1 CLN Ͻ14Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ2 Ͻ2 Ͻ2 MLN Ͻ16Ͻ118354Ͻ3 Ͻ3 Ͻ3 Lungs 1 8 Ͻ1 19 2 1 75 125 25 Spleen Ͻ11Ͻ141Ͻ1385 NMS cpts-248 Ͻ2.0 (0/6) Ͻ1.7 (0/6) NALT Ͻ11Ͻ1 Ͻ1 61 32 50 67 100 CLN 2 11 2 Ͻ1 Ͻ1 Ͻ133Ͻ3 MLN 2 201 3 5 142 12 12 376 47 Lungs Ͻ1 573 11 2 210 50 3 243 53 Spleen Ͻ1 167 2 10 15 7 2 7 3 NMS None 5.1 (6/6) 5.4 (6/6) NALT Ͻ1 Ͻ1 Ͻ12Ͻ1 Ͻ1 1 13 13 CLN Ͻ13Ͻ1 Ͻ13Ͻ1134 MLN 1 Ͻ1 Ͻ1 20 300 40 Ͻ1342 Lungs 1 2 1 Ͻ1 1 1 560 799 640 Spleen 2 2 Ͻ1 3 4 1 58 95 26

a wt virus challenge was administered 28 days following immunization. b Total cells were used from each site except for the lungs, from which mononuclear cells were obtained by density gradient purification. Cells were pooled from five animals on day 0, and from three animals on subsequent days. c Numbers in parentheses indicate proportion of mice from which virus was detected. These animals represent separate, repeat groups of animals similar to a subset of three groups presented in the first experiment presented in Table 1. The Journal of Immunology 3915

Table V. Both CD4ϩ and CD8ϩ T lymphocytes contribute to resistance to replication of wt RSV challenge virus following passive/active immunization at a time when Abs are present only at very low levels

c Ϯ d Depleting Abs Day 4 Postchallenge RSV Titers (mean log10 PFU/g tissue SEM) Serum Administered at the Time and Fraction of Animals from Which Virus Was Detectable Infused on Immunizing of wt RSV A2 Challenge Day Ϫ1 Virusa on Day 28b NT Lungs

MISe RSV cpts-248 Anti-CD4 2.0 Ϯ 0 3/6 Ͻ1.7 0/6 Anti-CD8 2.4 Ϯ 0.3 2/6 1.9 Ϯ 0.2 1/6 Anti-CD4 ϩ anti-CD8 4.2 Ϯ 0.1 6/6 3.6 Ϯ 0.1 6/6 Control Ab Ͻ2.0 0/6 Ͻ1.7 0/6 NMSf RSV cpts-248 Anti-CD4 2.1 Ϯ 0.1 3/6 Ͻ1.7 0/6 Anti-CD8 2.8 Ϯ 0.1 6/6 Ͻ1.7 0/6 Anti-CD4 ϩ anti-CD8 3.4 Ϯ 0.2 6/6 Ͻ1.7 0/6 Control Ab Ͻ2.0 0/6 1.9 Ϯ 0.2 1/6 NMS wt PIV3 Anti-CD4 4.3 Ϯ 0.1 6/6 5.4 Ϯ 0.1 6/6 Anti-CD8 4.1 Ϯ 0.1 6/6 5.4 Ϯ 0.1 6/6 Anti-CD4 ϩ anti-CD8 4.0 Ϯ 0.1 6/6 5.5 Ϯ 0.1 6/6 Control Ab 4.5 Ϯ 0.1 6/6 5.5 Ϯ 0.1 6/6

a BALB/c mice under light anesthesia were infected i.n. with 106.5 PFU in a 100-␮l volume. b Ϫ Ϫ

One milligram of depleting Ab was administered i.p. to each mouse on days 25, 27, and 30 after primary infection (days 3, 1, and 2 of the challenge phase of the Downloaded from experiment). c Mice were challenged i.n. with 106.3 PFU wt virus in a 100-␮l volume. d The values of six animals tested were used to calculate the mean. If virus was not detected in an individual animal, that animal was assigned a titer corresponding to the lower limit of detection for purposes of calculation of the mean for that tissue (2.0 log10PFU/g for NT and 1.7 log10PFU/g for lungs). e BALB/c mouse RSV-immune serum, administered i.p. f BALB/c mouse RSV-nonimmune serum, administered i.p. http://www.jimmunol.org/ lower than that in the primary infection group in which virus rep- However, in passive/active immunized mice (the MIS-treated/ lication was not restricted. Thus, an accelerated or augmented AFC cpts-248 group in Table V), T cell depletion had a major effect on response was not seen in the passive/active immunization group, protective efficacy in both the upper and lower respiratory tract. suggesting that Abs were not the principal mediators of resistance CD4ϩ or CD8ϩ T cell depletion alone had little effect on protec- to challenge in this group. Interestingly, the AFC that were in- tive efficacy in the NT or lungs. In contrast, simultaneous depletion duced in the passive/active group appeared in the MLN by day 4 of both CD4ϩ and CD8ϩ lymphocytes completely abrogated pro- then redistributed to the lungs 3 days later. tection in the NT and allowed a 100-fold higher level of replication in the lungs. It appears that CD4ϩ and CD8ϩ T cells each make Cell-mediated immunity studies independent contributions to this immunity. These data suggest by guest on September 25, 2021 ϩ ϩ Because neither humoral Abs nor AFC appeared to be the medi- that CD4 and CD8 T cells are the major mediators of resistance ators of resistance to replication of RSV challenge virus in the to challenge in the NT of passive/active immunized mice, whereas passive/active immunization group, we considered the possibility T cells and possibly a low level of Abs or other humoral factors that T cells are the principal mediators of resistance in this group. protect the lower respiratory tract in passive/active immunization. To test this hypothesis, passive/active immunized mice were de- The mediator of resistance accounting for the residual 90-fold re- ϩ ϩ pleted of T cells at the time of RSV challenge leaving humoral striction of replication in lungs in CD4 /CD8 T cell depleted immune mechanisms, if present, intact to restrict replication of passive/active immunized mice was not clear; however, we sug- RSV challenge virus. Mice were pretreated with MIS or NMS and gest that the low level of AFC (Table IV) detected in lung may then were infected with the RSV cpts-248 vaccine virus or a het- contribute to this effect. erologous wt respiratory virus (PIV3). At the time of wt RSV challenge 28 days after immunization, CD4ϩ, CD8ϩ,orCD4ϩ and CD8ϩ T cells were depleted in vivo using depleting Abs (Table Discussion V). As expected, replication of RSV challenge virus during pri- Immunization of very young infants against disease caused by mary infection (in the NMS-treated/PIV3 group) was not affected RSV will be difficult because of the need to immunize in the pres- by the depletion of T cells. ence of maternally acquired RSV Abs and because of the immu- Mice previously infected with cpts-248 in the absence of RSV- nological immaturity of the young infant. Passively acquired Abs specific passively acquired Abs (the NMS-treated/cpts-248 group suppress the primary Ab response to immunization with live RSV in Table V) would have developed both RSV-specific Abs (shown vaccine candidates, RSV subunit vaccines, or vaccinia-RSV re- in Tables II and III) as well as RSV-specific T cells, each of which combinant viruses (20, 21, 32). Indeed, suppression of infant hu- could mediate resistance to replication of RSV challenge virus. moral responses by maternal Abs has been demonstrated for a Protection of the NMS-treated/cpts-248 group in the lungs was wide variety of vaccines, including bacterial vaccines, such as per- most likely mediated by the high levels of Abs induced by immu- tussis (33) or tetanus vaccines (34) and viral vaccines such as those nization (as shown in Tables II-IV). Depletion of CD8ϩ and CD4ϩ for rabies virus (35), canine parvovirus (36), pseudorabies virus T cells resulted in a 20-fold increase in replication in the NT of (37, 38), foot and mouth disease virus (39), feline rhinotracheitis NMS-treated/cpts-248 animals, but no increase in the lungs of virus (40), and measles virus (41, 42). However, it was possible to these animals. This observation suggests that RSV Abs completely protect chimpanzees by immunization with live attenuated RSV protect the lungs, but local Abs plus local T cell immunity protect vaccine even when the induction of serum neutralizing Abs by the the nasopharynx. Abs present in the upper respiratory tract appear vaccine was suppressed by the passively acquired RSV Abs (21). to provide a 10-fold reduction in challenge virus replication, The mediators of resistance in this setting have not been previously whereas the remainder of the reduction requires T cells. defined, but identifying them should be very helpful for the design 3916 RSV Abs SUPPRESS HUMORAL BUT NOT T CELL-MEDIATED IMMUNITY of vaccination strategies that need to be implemented in the first humans, administration of a live attenuated RSV vaccine to 1-mo- weeks of life. old infants possessing maternally acquired RSV Abs induced a The current studies in mice show that live RSV vaccine candi- high level of resistance to replication of a second dose of vaccine dates can readily induce immunity even when immunization oc- virus given 1 mo later (50). Similar to the data presented here, very curs in the presence of physiologic levels of passively acquired few of those infants exhibited a serum RSV-specific IgG or neu- RSV neutralizing Abs. Although passive Ab suppresses the gen- tralizing Ab response following immunization in the presence of eration of serum and mucosal RSV-specific Abs and the RSV- RSV-specific maternal Abs. In contrast to passively/actively im- specific AFC response, a high level of immunity was seen in the munized mice, which failed to develop a systemic or mucosal Ab mice. As indicated above, passively acquired serum IgG Abs are response to immunization with live RSV vaccine, most human known to suppress the active IgG serum Ab response to immuni- infants possessing levels of maternally acquired RSV Abs similar zation, but much less is known about the effect of such passive Abs to those observed in mice in the present study, developed a mod- on the mucosal immune response to respiratory tract infection (43). erate level of serum and NW RSV IgA Abs. In these infants, the In the present study, the passively acquired RSV Abs suppressed resistance to replication of the second dose of RSV vaccine virus not only systemic Ab responses induced by immunization with live correlated with the presence of an Ab response to the RSV G attenuated RSV vaccines, but also suppressed the mucosal IgA, glycoprotein. Therefore, in humans the humoral immune response IgM, and IgG RSV-specific Ab responses as well as the regional may be of importance in the very young infant. It is unclear why and systemic Ab-secreting cell responses. These observations humans and mice differ in their responses to passive/active RSV identified a broad suppression of the humoral arm of the immune immunization in this regard. One factor may be the observation response and suggested that T cells contributed to the immunity that RSV infection of mice is only semipermissive, making it eas- Downloaded from observed in the passively/actively immunized mice. Indeed, CD4ϩ ier for a weak or a minor immune response to RSV to restrict its and CD8ϩ T cells each were found to make significant indepen- replication effectively upon reinfection. dent contributions to resistance to replication of RSV in those mice The mechanism of Ab-mediated immune suppression in human whose primary Ab response had been suppressed by passive Abs. neonates has not been fully defined. Passively acquired Abs may In such mice, humoral immunity also contributed to the restriction simply mediate blocking or accelerated clearance of immunizing of replication of RSV challenge virus in the lower respiratory tract, Ag by determinant-specific binding (51). Nevertheless, data from http://www.jimmunol.org/ whereas in the upper respiratory tract, the CD4ϩ and CD8ϩ T cells experimental systems suggest that Ag-Ab immune complexes were identified as the sole mediators of the observed resistance. formed in vivo can have potent immunoregulatory, including sup- The ability of CD4ϩ and CD8ϩ T cells induced by immunization pressive, effects, when such diverse Ags as pneumococcal poly- to restrict replication of RSV has been described previously (29, saccharides (52), Listeria monocytogenes organisms (53), and 44). At the other end of the spectrum, mice immunized with the model Ags like diphtheria and tetanus toxins, sheep red blood live RSV vaccine candidate in the absence of passively acquired cells, bacteriophages, and mammalian viruses are studied (54). RSV neutralizing Abs were also highly resistant to replication of Possible mechanisms of Ab-mediated immunoregulation include: 1) RSV challenge virus, but this resistance was only slightly dimin- Ab-Ag immune complex inhibition of B cell receptor signaling by ished by depletion of CD4ϩ and CD8ϩ T cells at the time of coligation of the receptor for the Fc region of IgG (FcRIIB1) and the by guest on September 25, 2021 challenge, indicating that resistance was mediated by Abs. Be- B cell receptor (55), and 2) inhibition of cytokine-mediated induction cause a high level of resistance to replication of RSV challenge of major histocompatibility complex expression in APC (56). virus was seen in passively/actively immunized mice over a 10- When primary Ab responses are suppressed by passive Abs, fold range of passively acquired RSV Ab concentrations, it appears priming of the B cell repertoire for secondary responses of greater that the relative contribution of the humoral and cellular arms of magnitude and quality (i.e., a memory response with enhanced the immune response to overall immunity varies depending on the neutralizing activity) may still occur. This observation was made extent of suppression of the primary Ab response by the passively previously in RSV passive/active immunization studies in chim- acquired RSV neutralizing Abs. panzees and bovines (21, 57, 58). Inhibition of primary Ab re- It was somewhat surprising to identify CD4ϩ and CD8ϩ T cells sponse with sparing of the secondary response has also been noted as major mediators of resistance to reinfection with RSV. T cells in experimental systems using Ag-Ab complexes, for example, are important for resolution of acute RSV infection (45). However, diphtheria toxin-antitoxin precipitates (54). Whether this phenom- they have been identified infrequently as contributors to protection enon also occurs in humans has not been adequately evaluated. against reinfection with RSV, and the resistance mediated by RSV- The unusual phenomenon of significant enhancement of secondary specific CD8ϩ T cells appears short-lived (2). The T cell-mediated Ab responses by prior passive/active immunization that we previ- mechanism of resistance identified in this paper may apply to other ously noted in chimpanzees (21) was not seen in the present study virus infections of infants who require immunization in the pres- in the mouse. In the primate studies, the primary serum Ab re- ence of maternal Abs, such as measles virus and influenza virus sponse to live RSV i.n. immunization as measured by ELISA bind- (41, 46, 47). Such studies with other viruses taken together with ing or neutralizing Abs was decreased at least 2-fold. Surprisingly, our data suggest that immunization with live respiratory viruses the secondary Ab response to challenge 1 mo following passive/ mediate protection by a novel T cell mechanism requiring the co- active immunization was greatly enhanced both in quantity (abso- operation of CD4ϩ and CD8ϩ T cells when the usual mechanism lute titer) and quality (ratio of neutralizing to binding Ab recipro- of protection, i.e., neutralizing Abs, is suppressed. Because the cal titer). The mechanism underlying this heightened secondary Ab duration of resistance against a respiratory virus mediated by ef- response in primates remains unexplained. There is no published fector T cells is generally of short duration (2, 48, 49), i.e., mea- data to suggest that RSV infection of human infants in the presence sured in weeks or months rather than years, it is likely that effec- of passively acquired RSV Abs primes for enhanced secondary tive durable immunity in the neonatal period will require responses. In this respect, the mouse studies reported here may readministration of vaccine until a sustained serum and mucosal better reflect the dynamics of immune response in humans infected Ab response is induced. in the presence of passively acquired Abs. One needs to exercise caution in extrapolating the findings from These studies in mice demonstrate that passively acquired RSV the present study in mice to the analogous situation in humans. In Abs suppress both systemic and local Ab responses to primary The Journal of Immunology 3917 infection with live attenuated RSV vaccine candidates. However, and A antibody response of infants and children to respiratory syncytial virus F this Ab-mediated immune suppression does not abrogate the de- and G glycoproteins following primary infection. J. Clin. Microbiol. 23:1009. 20. Murphy, B. R., R. A. Olmsted, P. L. Collins, R. M. Chanock, and G. A. Prince. velopment of resistance to wt virus challenge 1 mo later, which 1988. Passive transfer of respiratory syncytial virus (RSV) antiserum suppresses was shown to be mediated by both CD4ϩ and CD8ϩ T cells. De- the immune response to the RSV fusion (F) and large (G) glycoproteins expressed spite inhibition of the primary humoral response, passive Abs gen- by recombinant vaccinia viruses. J. Virol. 62:3907. 21. Crowe, J. E., Jr., P. T. Bui, G. R. Siber, W. R. Elkins, R. M. Chanock, and erally did not affect priming for a secondary Ab response following B. R. Murphy. 1995. Cold-passaged, temperature sensitive mutants of human challenge. Successful local and systemic immunity has been in- respiratory syncytial virus (RSV) are highly attenuated, immunogenic, and pro- tective in seronegative chimpanzees, even when RSV antibodies are infused duced in young infants with maternal Abs who were immunized shortly before immunization. Vaccine 13:847. with other live vaccines such as live poliovirus or live rotavirus 22. Crowe, J. E., Jr., P. L. Collins, W. T. London, R. M. Chanock, and B. R. Murphy. vaccines, but a multidose schedule of administration is required to 1993. A comparison in chimpanzees of the immunogenicity and efficacy of live attenuated respiratory syncytial virus (RSV) temperature-sensitive mutant vac- achieve optimal immunization. Our studies suggest that an effec- cines and vaccinia virus recombinants that express the surface glycoproteins of tive immunization strategy for RSV in young human infants who RSV. Vaccine 11:1395. possess maternal RSV Abs likely also will require multiple doses 23. Lewis, F. A., M. L. Rae, N. I. Lehman, and A. A. Ferris. 1961. A syncytial virus associated with epidemic disease of the lower respiratory tract in infants and of vaccine early in life. young children. Med. J. Australia 2:932. 24. Crowe, J. E., Jr., P. T. Bui, W. T. London, A. R. Davis, P. P. Hung, Acknowledgments R. M. Chanock, and B. R. Murphy. 1994. Satisfactorily attenuated and protective mutants derived from a partially attenuated cold passaged respiratory syncytial We thank Dr. Robert M. Chanock for critical review of the manuscript. virus mutant by introduction of additional attenuating mutations during chemical mutagenesis. Vaccine 12:691.

References 25. Crowe, J. E., Jr, P. T. Bui, A. R. Davis, R. M. Chanock, and B. R. Murphy. 1994. Downloaded from 1. Collins, P. L., K. McIntosh, and R. M. Chanock. 1996. Respiratory syncytial A further attenuated derivative of a cold-passaged temperature-sensitive mutant virus. In Fields Virology, B.N. Fields, D.M. Knipe, P.M. Howley, and of human respiratory syncytial virus (RSV cpts-248) retains immunogenicity and R.M. Chanock, eds. Lippincott Raven Press, New York, p. 1313. protective efficacy against wild-type challenge in seronegative chimpanzees. Vac- 2. Connors, M., P. L. Collins, C. Y. Firestone, and B. R. Murphy. 1991. Respiratory cine 12:783. syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to 26. Murphy, B. R., A. V. Sotnikov, L. A. Lawrence, S. M. Banks, and G. A. Prince. RSV challenge, but resistance induced by M2 and N proteins is relatively short- 1990. Enhanced pulmonary histopathology is observed in cotton rats immunized lived. J. Virol. 65:1634. with formalin inactivated respiratory syncytial virus (RSV) or purified F glyco- protein and challenged with RSV 3–6 months after immunization. Vaccine 8:497.

3. Liang, S., K. Mozdzanowska, G. Palladino, and W. Gerhard. 1994. Heterosub- http://www.jimmunol.org/ typic immunity to influenza type A virus in mice: effector mechanisms and their 27. Walsh, E. E., M. W. Brandriss, and J. J. Schlesinger. 1985. Purification and longevity. J. Immunol. 152:1653. characterization of the respiratory syncytial virus fusion protein. J. Gen. Virol. 4. Bachmann, M. F., T. M. Kundig, H. Hengartner, and R. M. Zinkernagel. 1997. 66:409. Protection against immunopathological consequences of a viral infection by ac- 28. Coates, H. V., D. W. Alling, and R. M. Chanock. 1966. An antigenic analysis of tivated but not resting cytotoxic T cells: T cell memory without “memory T respiratory syncytial virus isolates by a plaque reduction neutralization test. cells”? Proc. Natl. Acad. Sci. USA 94:640. Am. J. Epidemiol. 83:299. 5. Crowe, J. E., Jr. 1998. Immune responses of infants to infection with respiratory 29. Connors, M., A. B. Kulkarni, P. L. Collins, C. Y. Firestone, K. L. Holmes, viruses and live attenuated respiratory virus candidate vaccines. Vaccine 16:1423. H. C. Morse, III, and B. R. Murphy. 1992. Resistance to respiratory syncytial 6. Crowe, J. E., Jr. 1999. Host responses to respiratory virus infection and immu- virus (RSV) challenge induced by infection with a vaccinia virus recombinant nization. Curr. Top. Microbiol. Immunol. 236:191. expressing the RSV M2 protein (Vac-M2) is mediated by CD8ϩ T cells, while 7. Glezen, W. P., A. Paredes, J. E. Allison, L. H. Taber, and A. L. Frank. 1981. Risk that induced by Vac-F or Vac-G recombinants is mediated by antibodies. J. Virol.

of respiratory syncytial virus infection for infants from low-income families in 66:1277. by guest on September 25, 2021 relationship to age, sex, ethnic group, and maternal antibody level. J. Pediatr. 30. Connors, M., A. B. Kulkarni, C. Y. Firestone, K. L. Holmes, H. C. Morse, III, 98:708. A. V. Sotnikov, and B. R. Murphy. 1992. Pulmonary histopathology induced by 8. Glezen, W. P., A. L. Frank, L. H. Taber, and J. A. Kasel. 1984. Parainfluenza respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immu- virus type 3: seasonality and risk of infection and reinfection in young children. nized BALB/c mice is abrogated by depletion of CD4ϩ T cells. J. Virol. 66:7444. J. Infect. Dis. 150:851. 31. Epstein, S. L., J. A. Misplon, C. M. Lawson, E. K. Subbarao, M. Connors, and ␤ 9. Puck, J. M., W. P. Glezen, A. L. Frank, and H. R. Six. 1980. Protection of infants B. R. Murphy. 1993. 2-microglobulin-deficient mice can be protected against from infection with influenza A virus by transplacentally acquired antibody. J. In- influenza A infection by vaccination with vaccinia-influenza recombinants ex- fect. Dis. 142:844. pressing hemagglutinin and neuraminidase. J. Immunol. 150:5484. 10. Groothuis, J. R., E. A. Simoes, M. J. Levin, C. B. Hall, C. E. Long, 32. Murphy, B. R., G. A. Prince, P. L. Collins, S. W. Hildreth, and P. R. Paradiso. W. J. Rodriguez, J. Arrobio, H. C. Meissner, D. R. Fulton, and R. C. Welliver. 1991. Effect of passive antibody on the immune response of cotton rats to purified 1993. Prophylactic administration of respiratory syncytial virus immune globulin F and G glycoproteins of respiratory syncytial virus (RSV). Vaccine 9:185. to high-risk infants and young children. The Respiratory Syncytial Virus Immune 33. van Savage, J., M. D. Decker, K. M. Edwards, S. H. Sell, and D. T. Karzon. 1990. Globulin Study Group. N. Engl. J. Med. 329:1524. Natural history of pertussis antibody in the infant and effect on vaccine response. 11. IMpact-RSV Study Group. 1998. Palivizumab, a humanized respiratory syncytial J. Infect. Dis. 161:487. virus monoclonal antibody, reduces hospitalization from respiratory syncytial 34. Sarvas, H., S. Kurikka, I. J. Seppala, P. H. Makela, and O. Makela. 1992. Ma- virus infection in high-risk infants. Pediatrics 102:531. ternal antibodies partly inhibit an active antibody response to routine tetanus 12. Walsh, E. E., J. J. Schlesinger, and M. W. Brandriss. 1984. Protection from toxoid immunization in infants. J. Infect. Dis. 165:977. respiratory syncytial virus infection in cotton rats by passive transfer of mono- 35. Xiang, Z. Q., and H. C. Ertl. 1992. Transfer of maternal antibodies results in clonal antibodies. Infect. Immun. 43:756. inhibition of specific immune responses in the offspring. Virus Res. 24:297. 13. Taylor, G., E. J. Stott, M. Bew, B. F. Fernie, P. J. Cote, A. P. Collins, M. Hughes, 36. Hoare, C. M., P. DeBouck, and A. Wiseman. 1997. Immunogenicity of a low- and J. Jebbett. 1984. Monoclonal antibodies protect against respiratory syncytial passage, high-titer modified live canine parvovirus vaccine in pups with mater- virus infection in mice. Immunology 52:137. nally derived antibodies. Vaccine 15:273. 14. Prince, G. A., V. G. Hemming, R. L. Horswood, and R. M. Chanock. 1985. Immunoprophylaxis and immunotherapy of respiratory syncytial virus infection 37. Vannier, P. 1985. Experimental infection of fattening pigs with pseudorabies in the cotton rat. Virus Res. 3:193. (Aujeszky’s disease) virus: efficacy of attenuated live- and inactivated-virus vac- 15. Prince, G. A., V. G. Hemming, R. L. Horswood, P. A. Baron, and R. M. Chanock. cines in pigs with or without passive immunity. Am. J. Vet. Res. 46:1498. 1987. Effectiveness of topically administered neutralizing antibodies in experi- 38. Bouma, A., M. C. De Jong, and T. G. Kimman. 1997. The influence of maternal mental immunotherapy of respiratory syncytial virus infection in cotton rats. immunity on the transmission of pseudorabies virus and on the effectiveness of J. Virol. 61:1851. vaccination. Vaccine 15:287. 16. Mills, J. 5th, J. E. Van Kirk, P. F. Wright, and R. M. Chanock. 1971. Experi- 39. Francis, M. J., and L. Black. 1986. Response of young pigs to foot-and-mouth mental respiratory syncytial virus infection of adults: possible mechanisms of disease oil emulsion vaccination in the presence and absence of maternally de- resistance to infection and illness. J. Immunol. 107:123. rived neutralising antibodies. Res. Vet. Sci. 41:33. 17. Watt, P. J., B. S. Robinson, C. R. Pringle, and D. A. Tyrrell. 1990. Determinants 40. Johnson, R. P., and R. C. Povey. 1985. Vaccination against feline viral rhinotra- of susceptibility to challenge and the antibody response of adult volunteers given cheitis in kittens with maternally derived feline viral rhinotracheitis antibodies. experimental respiratory syncytial virus vaccines. Vaccine 8:231. J. Am. Vet. Med. Assoc. 186:149. 18. Murphy, B. R., D. W. Alling, M. H. Snyder, E. E. Walsh, G. A. Prince, 41. Siegrist, C. A., C. Barrios, X. Martinez, C. Brandt, M. Berney, M. Cordova, R. M. Chanock, V. G. Hemming, W. J. Rodriguez, H. W. Kim, and B. S. Graham. J. Kovarik, and P. H. Lambert. 1998. Influence of maternal antibodies on vaccine 1986. Effect of age and preexisting antibody on serum antibody response of responses: inhibition of antibody but not T cell responses allows successful early infants and children to the F and G glycoproteins during respiratory syncytial prime-boost strategies in mice. Eur. J. Immunol. 28:4138. virus infection. J. Clin. Microbiol. 24:894. 42. Albrecht, P., F. A. Ennis, E. J. Saltzman, and S. Krugman. 1977. Persistence of 19. Murphy, B. R., B. S. Graham, G. A. Prince, E. E. Walsh, R. M. Chanock, maternal antibody in infants beyond 12 months: mechanism of measles vaccine D. T. Karzon, and P. F. Wright. 1986. Serum and nasal-wash immunoglobulin G failure. J. Pediatr. 91:715. 3918 RSV Abs SUPPRESS HUMORAL BUT NOT T CELL-MEDIATED IMMUNITY

43. Yamazaki, H., H. Tsutsumi, K. Matsuda, K. Nagai, P. L. Ogra, and S. Chiba. 50. Wright, P. F., R. A. Karron, R. B. Belshe, J. Thompson, J. E. Crowe, Jr., 1994. Effect of maternal antibody on IgA antibody response in nasopharyngeal T. G. Boyce, L. L. Halburnt, G. W. Reed, S. S. Whitehead, R. Casey, et al. 2000. secretion in infants and children during primary respiratory syncytial virus in- Evaluation of a live, cold-passaged temperature sensitive, respiratory syncytial fection. J. Gen. Virol. 75:2115. virus (RSV) vaccine in infancy. J. Infect. Dis. 182:1331. 44. Plotnicky-Gilquin, H., A. Robert, L. Chevalet, J.-P. Haeuw, A. Beck, J.-Y. Bon- 51. Hutchinson, I. V., and H. Zola. 1978. Antigen-reactive cell opsonization: a mech- nefoy, C. Brandt, C.-A. Siegrist, T. N. Nguyen, and U. F. Power. 2000. CD4ϩ anism of antibody-mediated immune suppression. Cell. Immunol. 36:161. T-cell-mediated antiviral protection of the upper respiratory tract in BALB/c mice 52. Lee, C. J., Y. Takaoka, and T. Saito. 1987. Maternal immunization and the im- following parenteral immunization with a recombinant respiratory syncytial virus mune response of neonates to pneumococcal polysaccharides. Rev. Infect. Dis. G protein fragment. J. Virol. 74:3455. 9:494. 45. Graham, B. S. 1996. Immunological determinants of disease caused by respira- 53. Virgin, H. W. 4th, G. F. Wittenberg, G. J. Bancroft, and E. R. Unanue. 1985. tory syncytial virus. Trends Microbiol. 4:290. Suppression of immune response to Listeria monocytogenes: mechanism(s) of 46. Pertmer, T. M., A. E. Oran, J. M. Moser, C. A. Madorin, and H. L. Robinson. immune complex suppression. Infect. Immun. 50:343. 2000. DNA vaccines for influenza virus: differential effects of maternal antibody 54. Uhr, J. W., and G. Moller. 1968. The regulatory effect of antibody on the immune on immune responses to hemagglutinin and nucleoprotein. J. Virol. 74:7787. response. Adv. Immunol. 8:81. 47. Epstein, S. L., A. Stack, A., J. A. Misplon, C. Y. Lo, H. Mostowski, J. Bennink, 55. D’Ambrosio, D., K. L. Hippen, S. A. Minskoff, I. Mellman, G. Pani, and K. Subbarao. 2000. Vaccination with DNA encoding internal proteins of K. A. Siminovitch, and J. C. Cambier. 1995. Recruitment and activation of influenza virus does not require CD8ϩ cytotoxic T lymphocytes: either CD4ϩ or PTP1C in negative regulation of antigen receptor signaling by Fc␥RIIB1. Science CD8ϩ T cells can promote survival and recovery after challenge. Int. Immunol. 268:293. 12:91. 56. Virgin, H. W. 4th, G. F. Wittenberg, and E. R. Unanue. 1985. Immune complex 48. Kulkarni, A. B., P. L. Collins, I. Bacik, J. W. Yewdell, J. R. Bennink, J. E. Crowe, effects on murine macrophages. I. Immune complexes suppress interferon-␥ in- Jr., and B. R. Murphy. 1995. Cytotoxic T cells specific for a single peptide on the duction of Ia expression. J. Immunol. 135:3735. M2 protein of respiratory syncytial virus are the sole mediators of resistance 57. Kimman, T. G., F. Westenbrink, and P. J. Straver. 1989. Priming for local and induced by immunization with M2 encoded by a recombinant vaccinia virus. systemic antibody memory responses to bovine respiratory syncytial virus: effect J. Virol. 69:1261. of amount of virus, virus replication, route of administration and maternal anti- 49. Tao, T., F. Davoodi, C. J. Cho, M. H. Skiadopoulos, A. P. Durbin, P. L. Collins, bodies. Vet. Immunol. Immunopathol. 22:145. and B. R. Murphy. 2000. A live attenuated recombinant chimeric parainfluenza 58. Kimman, T. G., F. Westenbrink, B. E. Schreuder, and P. J. Straver. 1987. Local Downloaded from virus (PIV) candidate vaccine containing the hemagglutinin-neuraminidase and and systemic antibody response to bovine respiratory syncytial virus infection fusion glycoproteins of PIV1 and the remaining proteins from PIV3 induces and reinfection in calves with and without maternal antibodies. J. Clin. Microbiol. resistance to PIV1 even in animals immune to PIV3. Vaccine 18:1359. 25:1097. http://www.jimmunol.org/ by guest on September 25, 2021