Quantitative relationship between anticapsular antibody measured by enzyme-linked immunosorbent assay or radioimmunoassay and protection of mice against challenge with Streptococcus pneumoniae serotype 4.

We have recently shown that a substantial proportion of antibody to pneumococcal polysaccharide as measured by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay is removed by adsorption with pneumococcal cell wall polysaccharide (CWPS). The present study was undertaken to validate the hypothesis that only serotype-specific antibody that remains after adsorption with CWPS provides protection against pneumococcal infection. Serum samples were obtained from human subjects before and after they had been vaccinated with pneumococcal polysaccharide vaccine. Antibody to Streptococcus pneumoniae serotype 4 was measured by ELISA without adsorption or after adsorption of serum with CWPS. Groups of mice were injected with graded doses of serum and then challenged intraperitoneally with 10, 100, or 1,000 50% lethal doses (LD50) of S. pneumoniae serotype 4. Without adsorption, prevaccination sera from five healthy adults appeared to contain up to 33 micrograms of antibody to S. pneumoniae serotype 4 antigen per ml; adsorption with CWPS removed all detectable antibody, and pretreating mice with up to 0.1 ml of these sera (less than or equal to 3.3 micrograms of antibody) failed to protect them against challenge with 100 LD50. In contrast, postvaccination sera contained 2.9 to 30 micrograms of antibody per ml that was not removed by adsorption. Diluting sera to administer desired amounts of serotype-specific immunoglobulin G showed a significant relationship between protection and antibody remaining after adsorption (P less than 0.05 by linear regression analysis); 150 ng was uniformly protective against 1,000 LD50, and 50 ng was protective against 100 LD50. These studies have, for the first time, quantitated the amount of serotype-specific antibody that protects mice against challenge with S. pneumoniae type 4. In light of these observations, it is necessary to reassess current concepts regarding the presence of antipneumococcal antibody in the unvaccinated population, responses to pneumococcal vaccination, and protective levels of immunoglobulin G.     

PspA, a surface protein of Streptococcus pneumoniae, is capable of eliciting protection against pneumococci of more than one capsular type.

Monoclonal antibodies against pneumococcal surface protein A (PspA) have been shown to protect mice from fatal pneumococcal infection. PspA is highly variable serologically, raising the possibility that PspA from one strain might not be able to elicit protective responses against strains which possess serologically different PspA. We have prepared a lambda gt11 library of pneumococcal genomic DNA and identified a clone expressing PspA. The recombinant PspA in this phage lysate elicited protection against pneumococcal infections with three strains of two different capsular serotypes. This finding demonstrated that PspA could elicit a protective response in the absence of other pneumococcal antigens. The observed protection was probably antibody mediated because it could be passively transferred with immune sera. Lambda lysates producing pneumococcal proteins other than PspA failed to elicit protection against fatal pneumococcal infection.     

Evaluation of the role of the pneumococcal Forssman antigen (F-polysaccharide) in the cross-serotype protection induced by pneumococcal subcellular preparations.

We tested the hypothesis that the capacity of subcellular preparations of rough pneumococci to give cross-serotype protection is due to the presence of the pneumococcal Forssman antigen (F-polysaccharide). We found by hemagglutination inhibition that the Forssman antigen is present in the subcellular extracts. However, we concluded that the Forssman antigen is not the protective immunogen in the pneumococcal subcellular preparation, since absorption with sheep erythrocytes failed to remove the protective capacity from antiserum raised against the vaccine. Other evidence mitigating against the pneumococcal Forssman antigen being the protective immunogen included the absence of a detectable hemolytic titer in protective antiserum raised against the subcellular preparation, the failure of high-titered sheep hemolysin to passively protect mice against pneumococcal infection, and the failure of purified F-polysaccharide to immunize mice against pneumococcal infection.     

Host defenses in murine malaria: analysis of the mechanisms of immunity to Plasmodium berghei generated in response to immunization with formalin-killed blood-stage parasites.

Syngeneic B6D2F1 (C57Bl/6 x DBA/2) mice were immunized with a nonliving antigen prepared from mixed blood forms of Plasmodium berghei strain NYU-2. Consistently greater than 80% of the vaccinated mice survived virulent challenge, and protective immunity was demonstrable from 1 week through at least 4 months after immunization. However, vaccination did not prevent the development of patient infection after challenge. Instead, infections in vaccinated mice progressed to about 10% parasitemia and were then subsequently cleared. In contrast, infections initiated in nonvaccinated mice progressed beyond 10% parasitemia and were uniformly fatal within 4 weeks. Sera collected from normal mice, nonvaccinated mice infected with P. berghei, or vaccinated mice before challenge failed to passively protect recipients against virulent infection. On the other hand, sera collected from vaccinated mice after recovery from a challenge infection conferred upon passively immunized recipients protection from homologous virulent challenge, which was manifest as a delay in the onset of overt infection. It was concluded, therefore, that vaccination altered the immunological potential of the host in such a way as to allow the production of a protective humoral factor, probably specific antibody, in response to infection with the virulent parasites.     

Relationship between surface accessibility for PpmA, PsaA, and PspA and antibody-mediated immunity to systemic infection by Streptococcus pneumoniae

Antibodies to capsular polysaccharide (PS) are protective against systemic infection by Streptococcus pneumoniae, but the large number of pneumococcal serogroups and the age-related immunogenicity of pure PS limit the utility of PS-based vaccines. In contrast, cell wall-associated proteins from different capsular serotypes can be cross-reactive and immunogenic in all age groups. Therefore, we evaluated three pneumococcal proteins with respect to relative accessibility to antibody, in the context of intact pneumococci, and their ability to elicit protection against systemic infection by encapsulated S. pneumoniae. Sequences encoding pneumococcal surface adhesin A (PsaA), putative protease maturation protein A (PpmA), and the N-terminal region of pneumococcal surface protein A (PspA) from S. pneumoniae strain A66.1 were cloned and expressed in Escherichia coli. The presence of genes encoding PsaA, PpmA, and PspA in 11 clinical isolates was examined by PCR, and the expression of these proteins by each strain was examined by Western blotting with antisera raised to the respective recombinant proteins. We used flow cytometry to demonstrate that PspA was readily detectable on the surface of the pneumococcal strains analyzed, whereas PsaA and PpmA were not. Consistent with these observations, mice with passively or actively acquired antibodies to PspA or type 3 PS were equivalently protected from homologous systemic challenge with type 3 pneumococci, whereas mice with passively or actively acquired antibodies to PsaA or PpmA were not effectively protected. These experiments support the hypothesis that the extent of protection against systemic pneumococcal infection is influenced by target antigen accessibility to circulating host antibodies.     

Genetic immunization with the region encoding the alpha-helical domain of PspA elicits protective immunity against Streptococcus pneumoniae

Pneumococcal surface protein A (PspA) is a pneumococcal virulence factor capable of eliciting protection against pneumococcal infection in mice. Previous studies have demonstrated that the protection is antibody mediated. Here we examined the ability of pspA to elicit a protective immune response following genetic immunization of mice. Mice were immunized by intramuscular injections with a eukaryotic expression vector encoding the alpha-helical domain of PspA/Rx1. Immunization induced a PspA-specific serum antibody response, and immunized mice survived pneumococcal challenge. Survival and antibody responses occurred in a dose-dependent manner, the highest survival rates being seen with doses of 10 microg or greater. The ability of genetic immunization to elicit cross-protection was demonstrated by the survival of immunized mice challenged with pneumococcal strains differing in capsule and PspA types. Also, immunized mice were protected from intravenous and intratracheal challenges with pneumococci. Similar to the results seen with immunization with PspA, the survival of mice genetically immunized with pspA was antibody mediated. There was no decline in the level of protection 7 months after immunization. These results support the use of genetic immunization to elicit protective immune responses against extracellular pathogens.     

Antipneumococcal effects of C-reactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens.

Antibodies to pneumococcal capsular polysaccharides are well known for their ability to protect against pneumococcal infection. Recent studies indicate that antibodies to cell wall antigens, including pneumococcal surface protein A and the phosphocholine (PC) determinant of teichoic acids as well as human C-reactive protein (which also binds to PC), can protect mice against pneumococcal infection. In the present study we compared the protective effects of these agents as measured by mouse protection, the blood bactericidal assay, and clearance of pneumococci from the blood and peritoneal cavity. Our findings extend previous results indicating that human C-reactive protein and antibodies to noncapsular antigens are generally less protective than anticapsular antibodies. The new results obtained indicate the following: (i) mouse protection studies with intraperitoneal and intravenous infections provide very similar results; (ii) monoclonal immunoglobulin G2a (IgG2a) antibodies to PC, like IgG1, IgG2b, and IgG3 antibodies to PC, are highly protective against pneumococcal infection in mice; (iii) human antibody to PC is able to protect against pneumococcal infection in mice; (iv) antibodies to PspA are effective at mediating blood and peritoneal clearance of pneumococci; (v) complement is required for the in vivo protective effects of both IgG and IgM antibodies to PC; (vi) IgG1, IgG2b, and IgG3 anti-PC antibodies all mediate complement-dependent lysis of PC-conjugated erythrocytes; and (vii) antibodies and human C-reactive proteins that are reactive with capsular antigens but not cell wall antigens are able to mediate significant antibacterial activity in the blood bactericidal assay.     

Immunological correlates for protection against intranasal challenge of Bacillus anthracis spores conferred by a protective antigen-based vaccine in rabbits

Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 x 10(5) conferred protection, whereas lower titers (between 10(4) and 10(5)) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.     

Pneumococcal pneumonia and bacteremia model in mice for the analysis of protective antibodies

Pneumococci cause infection by colonizing the nasopharynx and invading the mucosal surfaces. Infection models in mice, where the natural route of infection is mimicked, may be useful to study antibody mediated protection against pneumococcal pneumonia and bacteremia. We have established a pneumococcal pneumonia and bacteremia model in mice and investigated the protective capacity of human antibodies. Intranasal challenge with serotypes 1, 3, 6A and 8 caused lung infection and bacteremia which was lethal. Serotype 6B caused low, but detectable, infection and other serotypes tested were not virulent. Passive immunization with a human IgG preparation i.p. protected mice in a dose dependent manner against bacteremia caused by the virulent serotypes (except serotype 3) and partially or completely cleared pneumococci from the lungs of mice infected with serotypes 1, 6A and 8. Adsorption of antibodies with homologous capsular polysaccharides eliminated protection against disease but adsorption with cell wall polysaccharides (CWPS) did not. Furthermore, a good correlation was observed between protection of sera in vivo and opsonic activity in vitro. The results indicate that the model may be useful to analyse the levels, isotypes, specificity and other characteristics of human antibodies which protect against pneumococcal infection and to evaluate the protective potential of pneumococcal vaccine candidates.     

Protective properties and haemagglutinins in serum from humans and in serum from mice injected with a new polyvalent Pseudomonas vaccine.

Mice given single injections of a polyvalent pseudomonas vaccine produced anti-pseudomonas haemagglutinins against the 16 component immunogens of the multivalent vaccine. Mice passively immunized with sera from vaccinated mice were protected against lethal challenge by 8/10 strains of Ps. aeruginosa of homologous serotype. Protection by the serum was inversely proportional to the virulence of the challenge strains. Anti-pseudomonas haemagglutinins were always present in sera which passively protected mice against pseudomonas infection. Low levels of anti-pseudomonas haemagglutinins were present in some sera which failed to passively immunize mice against pseudomonas infection. Anti-pseudomonas haemagglutinins and antibodies involved in passive protection were mainly in the IgM fractions of mouse serum. Control human sera contained anti-pseudomonas haemagglutinins against most serotypes of Ps. aeruginosa. Sera from patients with burns contained high levels of anti-pseudomonas haemagglutinins against some but not all serotypes of Ps. aeruginosa. Sera from both controls and patients with burns passively protected mice against pseudomonas infection.     

Isotype responses to candidate vaccine antigens in protective sera obtained from mice vaccinated with irradiated cercariae of Schistosoma mansoni.

In experimental schistosomiasis, sera of mice multiply vaccinated with radiation-attenuated cercariae of Schistosoma mansoni passively transfer resistance against cercarial challenge to naive mice. To further characterize these sera, we tested their protective capacities in two mouse strains (C57BL/6J and CBA/J) and compared the antigen-specific isotype compositions of the different protective sera by means of the enzyme-linked immunosorbent assay. By using an array of purified schistosomal antigens, the patterns of antibody titers and isotypes differed for each experimental group and antigen. In the most-protective C57BL/6J sera, high levels of immunoglobulin G1 (IgG1), IgG2a, and IgG2b bound to heat shock protein 70 and the integral membrane protein Sm23, whereas recognition of these antigens by less-protective CBA/J sera was lower. Glutathione S-transferase (GST) was recognized predominantly by IgM antibodies of all vaccinated groups, and a significant portion of this response was directed against carbohydrate epitopes. Antibodies specific for triosephosphate isomerase, paramyosin, and Sm32 (hemoglobinase) were present in less-protective sera and thus seem less relevant for passive transfer of resistance. The results of this study suggest a contribution of IgG antibodies specific for heat shock protein 70 and Sm23, and possibly a contribution of GST-specific IgM antibodies, to the protective effect of sera from C57BL/6J mice vaccinated with irradiated cercariae.     

肺炎链球菌溶血素减毒突变体免疫小鼠可诱导抗体依赖的保护效应

目的 优化肺炎链球菌溶血素减毒突变体(△A146 Ply)蛋白小鼠免疫方案,评价其保护效果,并揭示△A146 Ply特异性抗体在保护中的作用.方法 分别使用△A146 Ply免疫小鼠1、2、3次,利用ELISA分析血清内特异性IgG滴度,效价测定1周后,使用肺炎链球菌D39菌株鼻腔攻毒评价3种免疫方式保护效力.通过肺内定植模型和败血症模型分析△A146 Ply对肺炎链球菌感染的保护效果.利用抗体吸附试验和模拟被动保护试验分析Ply特异性抗体的保护作用.结果 随着免疫次数的增加,小鼠血清内的IgG滴度显著升高.相比免疫两次的小鼠,3次免疫后的小鼠其体内的IgG升高超过20倍.在攻毒试验中,免疫3次后的小组接受同等剂量的D39攻毒,存活率最高,保护效率为50%.在定植模型中,△A146 Ply免疫后的小鼠,其肺内肺炎链球菌血清型19F的细菌量减少约50倍,14血清型菌减少超过20倍.在败血症模型中,使用1200 CFU的D39腹腔注射到免疫组及对照组小鼠体内,△A146 Ply免疫组有60%的小鼠能够抵抗这种致命剂量的D39感染,对照组小鼠全部死亡(P=0.0018).在被动保护试验中,Ply特异性抗体被吸附完全后的抗血清完全不能保护小鼠抵抗致死剂量的D39感染,60%的小鼠在接受抗体特异的抗血清后存活,两组存活率差异有统计学意义.结论 △A146 Ply能有效抵抗肺炎链球菌的感染,证实保护作用与其特异性抗体密切相关.     

A human monoclonal immunoglobulin M reduces bacteremia and inflammation in a mouse model of systemic pneumococcal infection

Antibody-based approaches to pneumococcal disease may hold promise for immunocompromised patients in whom vaccines are less immunogenic and/or in the context of antimicrobial resistance. Antibody-mediated protection against experimental pneumococcal pneumonia has been shown to depend on immunoregulation, but the relationship between antibody and protection against pneumococcal sepsis and immunoregulation has not been examined. Similarly, the requirement for B and T cells for antibody efficacy is not known. In this study, we determined the efficacy of the human pneumococcal capsular polysaccharide serotype 3-specific antibody, A7 (immunoglobulin M [IgM]), in secretory IgM (sIgM)(-/-), CD4(-/-), CD8(-/-), muMT(-/-), and SCID mice and investigated its effect on cytokine and chemokine expression in sera and spleens from mice with intact cellular immunity. A7 is known to be protective against systemic infection with serotype 3 and to require complement for efficacy. Compared to that of an isotype control antibody, A7 administration prolonged the survival of mice of each immunodeficient strain and was associated with a significant reduction in CFU in blood, lung, and spleen samples and a significantly reduced level of keratinocyte-derived chemokine (KC), interleukin-6 (IL-6), and macrophage inflammatory protein-2 (MIP-2) expression in normal and sIgM(-/-) mice. Studies with mice treated with penicillin revealed similar reductions in CFU and similar levels of IL-6, KC, or MIP-2 expression in A7- and penicillin-treated mice. These findings demonstrate that natural IgM and B and T cells are dispensable for A7-mediated protection against experimental pneumococcal sepsis and suggest that the efficacy of antibody-mediated protection depends on immunomodulation. Taken together, our data extend the association between antibody-mediated protection and immunomodulation to protection against systemic pneumococcal infection and to a clinically important serotype often responsible for pneumococcal sepsis.     

Antibodies against pneumococcal C-polysaccharide are not protective

The ability of antibodies against C-polysaccharide (C-Ps) to protect against experimental pneumococcal infection was examined in a mouse model. No protection against types 6A and 14 pneumococcal infection could be demonstrated neither with mouse monoclonal antibodies against C-Ps, specific for phosphorylcholine (PC) or for C-Ps backbone, nor for polyclonal rabbit immunsera against C-Ps. The monoclonal antibody with PC-specificity was protective against infection with type 27 pneumococcus, that has PC as part of its capsular polysaccharide. Type-specific mono- and polyclonal antibodies were highly protective against infection with the homologous type. We conclude that no species-specific protection can be achieved against intraperitoneal Streptococcus pneumoniae infection with optimally capsulated bacteria in outbred mice by passive immunization with antibodies to C-Ps.     

Resistance to meningococcemia apparently conferred by anti-H.8 monoclonal antibody is due to contaminating endotoxin and not to specific immunoprotection.

We evaluated the ability of a monoclonal antibody directed against the common H.8 antigen of pathogenic Neisseria sp. to confer passive protection against meningococcal disease in mice. The apparent protection conferred by antibody purified from tissue culture supernatant was actually the result of endotoxin contamination of buffers and tissue culture media. Endotoxin-free anti-H.8 antibody was not protective. The possibility of endotoxin contamination should be considered when evaluating immunity conferred by passively administered antibody in animal models.     

Heterogeneity of neutralization-related epitopes within a bluetongue virus serotype

Twenty-four monoclonal antibodies raised against a 1962 Wyoming isolate of blue-tongue virus serotype 17 (BTV 17) were tested against 20 other field isolates of this serotype in a solid-phase radioimmunoassay (RIA), neutralization, and mouse passive protection tests. Of the 21 antibodies that bound in RIA to acetone-fixed BTV-infected cells, 18 bound to cells infected with any of the BTV 17 isolates and 3 detected minor antigenic differences in two isolates. The remaining 3 antibodies, that bound in RIA only to unfixed virus-infected cells detected additional differences. Of the 3 antibodies binding to unfixed virus-infected cells one bound to all but 2 isolates. A second antibody, 6C3A.2, bound only to the Wyoming isolate and passively protected mice against this isolate. The third antibody, 6C2A.4.2, bound to the Wyoming isolate and to 8 isolates from the mid-South U. S., but not to 12 isolates from California. Antibody 6C2A.4.2 neutralized the Wyoming and mid-South isolates to which it bound and passively protected mice against the Wyoming isolate but provided little or no protection against 4 California isolates tested. Polyclonal serum from mice immunized against Wyoming BTV 17 bound in RIA to all BTV 17 isolates and neutralized all isolates. Thus, three neutralization-related antigenic determinants were disclosed, one (perhaps a set) recognized by immune sera on all BTV 17 isolates, a second recognized by antibody 6C2A.4.2 on the Wyoming and 8 mid-South isolates, and a third recognized by antibody 6C3A.2 only on the Wyoming isolate. These differences may be important in selection of virus strains for vaccine development.     

Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites.

Rhesus monkeys vaccinated with merozoites in FCA are protected against challenge with several strains and variants of Plasmodium knowlesi. Vaccination induces sterilizing immunity which is species specific. Merozoite-blocking (inhibitory) antibody usually correlates with clinical immunity and protection can be passively transferred with immune sera provided these contain high levels of inhibitory antibody. However, vaccination using adjuvants other than FCA may induce inhibitory antibody without clinical protection. In addition, vaccinated animals may become susceptible to challenge 4-5 weeks after splenectomy, although inhibitory antibody levels are not reduced. These observations indicate that immunity induced by merozoite vaccination involves: (i) merozoite blocking (inhibitory) antibody, (ii) specific antibody or immune complexes acting synergistically with cytotoxic splenic cells stimulated by FCA.     

Perforin and gamma interferon are critical CD8+ T-cell-mediated responses in vaccine-induced immunity against Leishmania amazonensis infection

Previous studies have demonstrated that protection against New World leishmaniasis caused by Leishmania amazonensis can be elicited by immunization with the developmentally regulated Leishmania amastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8(+) (as well as CD4(+)) effector responses is required for protection. Further, mice lacking beta(2)-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8(+) T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8(+) T cells, secreting gamma interferon (IFN-gamma) and expressing perforin, was observed at the site of infection; subsequently, activated CD4(+) T cells producing IFN-gamma were primarily found. As protection correlated with the ratio of total IFN-gamma-producing cells (CD4(+) and CD8(+) T cells) to macrophages found at the site of infection, a role for IFN-gamma was evident; in addition, vaccination of IFN-gamma-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8(+) T cell effector mechanisms (perforin, IFN-gamma) are clearly required in the protective immune response against L. amazonensis infection in vaccinated mice.     

Rabbit immunoglobulin responses to the flagella, somatic, and protective antigens of a highly protective strain of Clostridium chauvoei.

The immunoglobulin response of rabbits to the flagells (H), somatic (O), and protective antigens of a highly protective strain of Clostridium chauvoei was studied using antisera that had been fractionated by Sephadex G-200 chromatography. The H antigen elicited the characteristic agglutinin response to a protein antigen--early production of 19S globulin followed by persistent 7S globulin production. The O antigen stimulated a transient agglutinin response which was detected in both the 19S and 7S serum fractions. Protective antibody was assayed by passive protection tests in mice. Using these tests the protective activity of the rabbit sera was found to be confined exclusively to the 7S serum fractions. Purified immunoglobulin G, prepared by DEAE-cellulose chromatography of the above sera, was also tested and found to confer considerable passive protection on mice. It is considered that either the protective antigen fails to stimulate an immunoglobulin M response or that immunoglobulin M is relatively ineffective in conferring protection against infection in the mouse passive protection tests.     

Immune responses to labial infection of BALB/c mice with herpes simplex virus type 1.

The kinetics of appearance of five humoral antibody responses (micro-neutralization assay [NT], complement fixation [CF], enzyme-linked immunosorbent assay [ELISA], radioimmunoassay [RIA], antibody-dependent cell-mediated cytotoxicity [ADCC]), were compared during labial infection of BALB/c mice with herpes simplex virus type 1 strain Patton. The ELISA/RIA antibody responses were present in most mice by day 5 after infection, at the beginning of the herpetic lip lesions; antibody effective in ADCC showed identical early kinetics. In contrast, NT/CF antibodies were not detected in most mice until day 10, at the time of resolution of the herpetic lip lesions. The humoral immune responses persisted for at least 6 months after infection. The NT and CF responses were closely correlated in time of appearance and titers (r = 0.9), as were the ELISA and RIA responses (r = 0.99). However, there was little correlation between NT/CF and ELISA/RIA responses (r = 0.02). The kinetics of the delayed type hypersensitivity response showed similar kinetics of appearance to the ELISA/RIA/ADCC humoral responses, and peaked similarly, but waned gradually over 2 months. The importance of antibody in protection against labial herpes simplex virus type 1 infection was demonstrated by the ability of passively transferred convalescent serum (that produced a minimum NT titer of 10 in recipient mice) to protect against development of herpetic lesions and death.