Listeria Pneumonitis: Influence of Route of Immunization on Resistance to Airborne Infection

Mice that are immunized with an airborne inoculum of BCG are more highly resistant to airborne challenge with Mycobacterium tuberculosis than are mice that are immunized by the subcutaneous or intravenous route. To discover whether this phenomenon is peculiar to tuberculosis, we studied the influence of the route of immunization upon pulmonary resistance in Listeria monocytogenes infection. Mice were immunized by the airborne, intravenous, or footpad route and were subsequently challenged by the same route at 1 to 4 weeks after immunization. Mice were highly and uniformly resistant to intravenous challenge, regardless of the route of immunization. The route of immunization bore no influence upon resistance to footpad infection, but resistance was appreciably better in mice challenged within 2 weeks of immunization than it was at later time points. In mice immunized by the footpad and intravenous routes, the pattern of resistance to airborne and footpad challenges was similar, in that there was substantially less immunity at 4 weeks than at 2 weeks after immunization. However, mice immunized by the airborne route were highly resistant to airborne challenge, regardless of the interval between immunization and reinfection. In this last respect, resistance of the lungs to reinfection was similar after Listeria and tuberculosis pneumonitis. It is suggested that a similar pattern of resistance may prevail in pneumonitis caused by other facultative intracellular parasites.     

Lymphocyte recruitment and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior Mycobacterium bovis BCG immunization

Mycobacterium tuberculosis infects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4(+) T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosol M. tuberculosis infection in mice occurs predominantly through the interaction of alpha(4)beta(1) integrin on CD4(+) T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization with Mycobacterium bovis BCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4(+) T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge with M. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4(+) T cells to the lung following primary immunization by the three routes. Expansion of CD44(hi) CD62L(low) CD4(+) T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4(+) T cells expressed alpha(4)beta(1) integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting alpha(4)beta(1)(+) CD4(+) T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of alpha(4)beta(1)(+) CD4(+) memory T cells occurred following M. tuberculosis challenge. The three routes of BCG immunization resulted in the same level of protection against aerosol M. tuberculosis or BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.     

Specific and nonspecific resistance in mice immunized with irradiated Myobacterium leprae.

Following subcutaneous inoculation of irradiated Mycobacterium leprae (I-ML) into the left hind footpad of mice, there was increased resistance to Listeria monocytogenes, indicative of macrophage activation, at the immunization site. In spite of the high level of localized macrophage activation which was proportioned to the immunizing dose of I-ML, no such activity could be demonstrated systemically in these mice, as evidenced by the absence of increased resistance to an intravenous challenge with L. monocytogenes. Under these conditions, I-ML-immunized mice were nonetheless resistant to intravenous infection with either M. tuberculosis or M. bovis BCG, and this immunity was transferred to normal recipients using spleen or lymph node cells. Neonatal thymectomy completely abolished the development of antimycobacterial immunity after vaccination with I-ML, but immunity was restored by an intraperitoneal infusion of syngeneic thymocytes. Systemic nonspecific resistance could be generated in I-ML-immunized mice by an intravenous injection of disrupted I-ML. This study reveals that, after subcutaneous vaccination with I-ML, there is local accumulation of activated macrophages at the inoculation site and a widespread distribution of lymphocytes which are sensitized to mycobacterial antigens. Nonspecific resistance is mediated by the former cells and specific antimycobacterial immunity by the latter.     

Mycobacterium bovis BCG induces similar immune responses and protection by rectal and parenteral immunization routes

We compared cellular immune responses to rectal, subcutaneous, and intradermal administration of Mycobacterium bovis BCG for 5 to 20 weeks in mice, guinea pigs, and macaques. Strong lymphoproliferative responses were induced in spleen cells after in vitro stimulation with purified protein derivative in guinea pigs and macaques, whatever the route of immunization. Comparable high numbers of gamma interferon- and tumor necrosis factor alpha-producing cells were found in the spleen after rectal, subcutaneous, and intradermal immunization of mice and macaques. Similar levels of precursors of cytotoxic T lymphocytes specific for mycobacterial antigens were observed in mice for all immunization routes. In macaques, cytotoxic activity, determined only at the end of the experiment (20 weeks), was similar after rectal and intradermal immunization. Six months after immunization, rectal and subcutaneous routes induced in mice similar levels of protective immunity against challenge with a virulent Mycobacterium tuberculosis strain (H37Rv). Rectal immunization gave immune responses and protective capacity similar to those for parenteral immunization and seemed to be a promising new route of vaccination against tuberculosis; in our study, immunization via the rectal route never induced side effects associated with parenteral routes (axillary adenitis) and could also effectively reduce the risks of viral transmission associated with unsafe injections in the developing world.     

Intranasal vaccination with the recombinant Listeria monocytogenes ΔactA prfA* mutant elicits robust systemic and pulmonary cellular responses and secretory mucosal IgA

We previously showed that recombinant (r) Listeria monocytogenes carrying ΔactA and a selected prfA* mutation (r-Listeria ΔactA prfA*) secreted >100-fold more immunogen in broth culture than wild-type r-Listeria or r-Listeria ΔactA and elicited much greater cellular and humoral immune responses than r-Listeria ΔactA after intravenous vaccination of mice. Here, we conducted comparative studies evaluating vaccine-elicited immune responses in systemic and mucosal sites after intranasal, intravenous, intraperitoneal, or subcutaneous immunization of mice with r-Listeria ΔactA prfA* vaccine candidates. Intranasal vaccination of mice with r-Listeria ΔactA prfA* vaccine candidates elicited a robust gamma interferon-positive (IFN-γ(+)) cellular response in systemic sites, although intravenous or intraperitoneal immunization was more efficient. Surprisingly, intranasal vaccination elicited an appreciable pulmonary IFN-γ(+) cellular response that was nonstatistically higher than the magnitude induced by the intravenous route but was significantly greater than that elicited by subcutaneous immunization. Furthermore, although intranasal r-Listeria ΔactA prfA* delivery induced poor systemic IgG responses, intranasal vaccination elicited appreciable secretory immunogen-specific IgA titers that were similar to or higher in mucosal fluid than those induced by subcutaneous and intravenous immunizations. Thus, intranasal vaccination with r-Listeria ΔactA prfA* appears to be a useful approach for eliciting robust systemic and pulmonary cellular responses and measurable secretory mucosal IgA titers.     

Immunization with Single Oral Dose of Alginate‐Encapsulated BCG Elicits Effective and Long‐Lasting Mucosal Immune Responses

Effective vaccination against pathogens, which enter the body through mucosal surfaces, requires the induction of both mucosal and systemic immune responses. Here, mucosal as well as systemic immune responses in the lung and spleen of BALB/c mice which were orally vaccinated with a single dose of alginate‐encapsulated bacille Calmette–Guerin (BCG) were evaluated. Twenty weeks after immunization, the vaccinated mice were challenged intranasally with BCG. Twelve weeks after immunization and 5 weeks after challenge, the immune responses were evaluated. Moreover, immune responses were compared with those of mice that were vaccinated with free BCG by subcutaneous (sc) and oral routes. Twelve weeks after the immunization, serum IgG level was higher in the sc‐immunized mice, while serum IgA level was higher in the orally immunized mice with encapsulated BCG. Significant productions of both IgG and IgA were only detected in lungs of mice orally immunized with encapsulated BCG. Proliferative and delayed‐type hypersensitivity responses and IFN‐γ production were significantly higher in mice immunized orally with encapsulated BCG, compared to mice immunized orally with free BCG. After challenge, the levels of IFN‐γ were comparable between sc‐immunized mice with free BCG and orally immunized with encapsulated BCG; however, significantly less IL‐4 was detected in mice which had received encapsulated BCG via oral route. Moreover, significant control of the bacilli growth in the lung of the immunized mice after intranasal challenge with BCG was documented in mice vaccinated with encapsulated BCG. These results suggest that oral immunization with alginate‐encapsulated BCG is an effective mean of inducing mucosal and systemic specific immune responses.     

Immune response after adjuvant mucosal immunization of mice with inactivated influenza virus

Satisfactory mucosal immunity in the respiratory tract is very important for protection against influenza. It can be achieved only by mucosal immunization. Mucosal vaccination with inactivated influenza virus may not be sufficiently effective and suitable adjuvants are therefore sought. We tested intratracheal immunization of mice with inactivate B type influenza virus in a mixture with formolized G+ bacterium Bacillus firmus, whose adjuvant effects have previously been documented in another system. The treatment resulted in a marked increase of both systemic and mucosal antibody response in IgG and IgA classes. Stimulation of T lymphocytes after adjuvant immunization was very mild, no proliferation taking place after specific stimulation with antigen in vitro. However, slightly increased systemic (spleen) and local (lungs) production of cytokines without perceptible Th1/Th2 polarization was determined. B. firmus is an efficient adjuvant in respiratory tract immunization while with subcutaneous immunization it lowers the antibody response.     

Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis

Whether the intranasal (i.n.) route of Mycobacterium bovis BCG vaccination provides better protection against pulmonary tuberculosis than subcutaneous (s.c.) vaccination remains an incompletely solved issue. In the present study, we compared both immune responses and protection elicited by single BCG vaccinations via the i.n. or s.c. route in BALB/c mice. While both i.n. and s.c. vaccination triggered comparable levels of primary immune activation in the spleen and draining lymph nodes, i.n. vaccination led to a greater antigen-specific gamma interferon recall response in splenocytes than s.c. vaccination upon secondary respiratory mycobacterial challenge, accompanied by an increased frequency of antigen-specific lymphocytes. There was also a quicker cellular response in the lungs of i.n. vaccinated mice upon mycobacterial challenge. Mice vaccinated i.n. were found to be much better protected, particularly in the lung, than s.c. vaccinated counterparts against pulmonary tuberculosis at both 3 and 6 months postvaccination. These results suggest that the i.n. route of vaccination improves the protective effect of the current BCG vaccine.     

Immune Response to Mycobacterium tuberculosis in Rats

After intravenous injection into rats, both the attenuated strain R1Rv and the virulent strain H37Rv of Mycobacterium tuberculosis grow in the liver and spleen. However, the infected rats mount a specific immune response with great rapidity, giving a false impression of natural resistance to the tubercle bacillus. Adoptive immunity to tuberculosis was achieved by transferring thoracic duct cells from immunized donors to normal syngeneic recipients. The transferred immunity was vested in a population of lymphocytes uncontaminated with macrophages. The adoptive immunity was effectively expressed against both attenuated and virulent tubercle bacilli, and it was shown to be immunologically specific. Lymphocytes which conferred immunity to tuberculosis were not protective against Listeria monocytogenes infection, and vice versa. Immunity could not be transferred with either normal thoracic duct lymphocytes (TDL), heat-killed sensitized TDL, or serum from specifically immunized donors. The ability of TDL from BCG-immunized donors to confer immunity was maintained at an unimpaired level for at least 3 months after immunization.     

Paucibacillary tuberculosis in mice after prior aerosol immunization with Mycobacterium bovis BCG

To develop a murine model of paucibacillary tuberculosis for experimental chemotherapy of latent tuberculosis infection, mice were immunized with viable Mycobacterium bovis BCG by the aerosol or intravenous route and then challenged six weeks later with virulent Mycobacterium tuberculosis. The day after immunization, the counts were 3.71 +/- 0.10 log(10) CFU in the lungs of aerosol-immunized mice and 3.65 +/- 0.11 and 4.93 +/- 0.07 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Six weeks later, the lungs of all BCG-immunized mice had many gross lung lesions and splenomegaly; the counts were 5.97 +/- 0.14 and 3.54 +/- 0.07 log(10) CFU in the lungs and spleens of aerosol-immunized mice, respectively, and 4.36 +/- 0.28 and 5.12 +/- 0.23 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Mice were then aerosol challenged with M. tuberculosis by implanting 2.37 +/- 0.13 log(10) CFU in the lungs. Six weeks after challenge, M. tuberculosis had multiplied so that the counts were 6.41 +/- 0.27 and 4.44 +/- 0.14 log(10) CFU in the lungs and spleens of control mice, respectively. Multiplication of M. tuberculosis was greatly limited in BCG-immunized mice. Six weeks after challenge, the counts were 4.76 +/- 0.24 and 3.73 +/- 0.34 log(10) CFU in the lungs of intravenously immunized and aerosol-immunized mice, respectively. In contrast to intravenously immunized mice, there was no detectable dissemination to the spleen in aerosol-immunized mice. Therefore, immunization of mice with BCG by the aerosol route prior to challenge with a low dose of M. tuberculosis resulted in improved containment of infection and a stable paucibacillary infection. This model may prove to be useful for evaluation of new treatments for latent tuberculosis infection in humans.     

Induction and suppression of cross-reactive antituberculosis immunity after Mycobacterium lepraemurium infection of mice.

Mice immunized with 10(8) live Mycobacterium lepraemurium in the footpad showed increased resistance to infection with BCG or M. tuberculosis R1Rv. This resistance could be transferred adoptively with lymphoid cells, signifying that the immunity was cross-reactive rather than nonspecific. Adoptive cross-reactive immunity to M. tuberculosis was also conferred by spleen cells from mice immunized with large doses of living or dead M. lepraemurium intravenously, a route of immunization that suppresses the induction of cell-mediated immunity to that organism. The presence of specific suppressor activity was sought in mice immunized intravenously with M. lepraemurium. It was found that mice preimmunized intravenously with living or dead M. lepraemurium and then infected with BCG did not confer levels of adoptive antituberculosis immunity as high as those conferred by mice immunized with BCG alone. Similarly, a mixture of BCG-sensitized and M. lepraemurium-sensitized cells did not convey as much immunity as BCG-sensitized cells alone, signifying suppression of the effector lymphocytes.     

Comparison of protective efficacy of subcutaneous versus intranasal immunization of mice with a Brucella melitensis lipopolysaccharide subunit vaccine

Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.     

Transfer of Adoptive Immunity to Tuberculosis in Mice

A system is described for studying adoptive immunity to tuberculosis in syngeneic mice. Donor mice were immunized with 10⁴ BCG intravenously, and lymphoid cells were harvested 28 days later. Adoptive immunity was measured in recipient mice in terms of the inhibition of growth of BCG in the liver and spleen following intravenous injection. Adoptive immunity was expressed optimally when recipients were sublethally irradiated (500 R), challenged with 10⁴ to 10⁵ viable organisms, and given sensitized lymphoid cells intravenously. Adoptive immunity was not manifest until 14 days after challenge and was effective against Mycobacterium tuberculosis H37Rv as well as BCG. Immunity could be conferred by spleen, lymph node, peritoneal exudate, and resident peritoneal (washout) cells. The lymphoid cells conferring immunity were shown to be thymus-dependent lymphocytes by virtue of their nonadherence to glass wool and sensitivity to anti-θ serum plus complement. The sensitized cells were relatively susceptible to both in vitro and in vivo X-irradiation.     

Studies on acquired systemic resistance in mice with oral immunization with Shigella sonnei

Virulent, viable Shigella sonnei bacilli introduced orally, induced in mice resistance against lethal intravenous (i.v.) infection with dysentery bacilli. The immunized animals survived lethal infection and also bacilli were effectively destroyed in the spleen and liver of the immunized animals. It was shown that orally induced immunity may be transferred into nonimmunized mice by means of serum and spleen cells taken on definite days after immunization of donors. Protective activity of serum appeared to be dependent on the presence of specific antibodies and was correlated with their titer. Protective activity of spleen cells occurred about 6 days after immunization of the cell donors with the bacilli. Cell suspensions, prepared from the spleens taken from donors on day 6, protected 100% of the recipients of these cells from lethal infection. Results of these studies indicate that bacterial infection developing in the intestine mobilizes besides local immunity also general immunity. Mechanism of the general immunity induced by orally administered dysentery bacilli is discussed.     

Effect of route of Mycobacterium bovis BCG administration on induction of suppression of sporozoite immunity in rodent malaria.

Intravenous immunization of mice with 16,000, 60Co--gamma-irradiated, attenuated sporozoites produced solid immunity to sporozoite-induced malaria when the mice were challenged 21 days after immunization. In contrast, mice injected by various routes with 10(7) viable units of Mycobacterium bovis (BCG) before immunization with irradiated sporozoites were not completely immune to challenge. The extent of reduced protection against viable sporozoites demonstrated with these animals was dependent upon the injection route mycobacteria. The intravenous administration of BCG induced the greatest degree of suppression, followed by the intraperitoneal and subcutaneous routes. BCG injected intramuscularly before sporozoite immunization did not suppress development of immunity. In contrast, mice injected with BCG after immunization with attenuated sporozoites exhibited a lesser degree of suppression. In these animals, only the intravenous injection of mycobacteria reduced immunity.     

Recruitment of lymphocytes to the lung through vaccination enhances the immunity of mice exposed to irradiated schistosomes.

The effector mechanism, which operates against challenge parasites in the lungs of C57BL/6 mice vaccinated once with irradiated cercariae of Schistosoma mansoni, is mediated by CD4+ T helper lymphocytes. However, adoptive transfer of immunity from vaccinated donors to naive recipients by using sensitized T cells has not proved successful. One explanation may be that the recruitment of sensitized T lymphocytes to the lungs by vaccinating parasites to arm that organ is not reproduced by transfer protocols. We have used the technique of parabiosis, as a means of adoptive transfer, to demonstrate the relevance of pulmonary T cells to protection. Sensitized and naive partners were joined surgically for a 28-day period, coincident with priming of the immune system. A vascular union rapidly developed, and sensitized T cells were detected in the spleens of the naive partners. When parabionts were challenged percutaneously 10 days after separation, the level of immunity transferred to the naive partners was approximately two-thirds that of their vaccinated counterparts. The naive partners, unlike the vaccinated animals, did not recruit lymphocytes to the lungs during the priming period. In contrast, after percutaneous challenge, schistosome-specific lymphocytes were recruited to the lungs of both separated parabionts. The importance of lymphocytes recruited to the lungs during the primary response was revealed by an intravenous challenge with lung schistosomula; this eliminates the opportunity for secondary immune responses prior to parasite arrival in the lungs. In this situation, the vaccinated partners showed 47% immunity while the naive partners were not protected. We conclude that the presence of specific T cells in the lungs at the time of challenge confers a significant advantage, permitting a more effective recall response than in animals lacking such resident cells.     

Gamma-irradiated scrub typhus immunogens: development of cell-mediated immunity after vaccination of inbred mice.

Mice immunized with three injections of gamma-irradiated Karp strain of Rickettsia tsutsugamushi were evaluated for the presence of cell-mediated immunity by using delayed-type hypersensitivity, antigen-induced lymphocyte proliferation, and antigen-induced lymphokine production. These animals also were evaluated for levels of circulating antibody after immunization as well as for the presence of rickettsemia after intraperitoneal challenge with viable Karp rickettsiae. After immunization with irradiated Karp rickettsiae, a demonstrable cell-mediated immunity was present as evidenced by delayed-type hypersensitivity responsiveness, lymphocyte proliferation, and production of migration inhibition factor and interferon by immune spleen lymphocytes. Also, a reduction in circulating rickettsiae was seen in mice immunized with irradiated rickettsiae after challenge with 1,000 50% mouse lethal doses of viable, homologous rickettsiae. All responses except antibody titer and reduction of rickettsemia were similar to the responses noted in mice immunized with viable organisms. Antibody levels were lower in mice immunized with irradiated rickettsiae than in mice immunized with viable rickettsiae. Furthermore, mice that were immunized with viable rickettsiae demonstrated markedly lower levels of rickettsemia after intraperitoneal challenge compared with either mice immunized with irradiated rickettsiae or nonimmunized mice.     

Transfer of immunity by means of spleen cells from mice immunized with outer membrane proteins of Shigella flexneri

Intraperitoneal immunization of mice with a single dose (5 micrograms) of outer membrane proteins (OMP) of Shigella flexneri was found to evoke in the spleen the appearance of cells by means of which immunity to lethal dose of Shigella could be transferred into other mice. Active cells capable of transferring immunity appeared in the spleen of the animals as early as on day 3, reached the strongest protective activity on day 4 and disappeared on day 8 after immunization. Active cells from animals immunized with two doses of OMP maintained in the spleens for 19 days. The experiments revealed that immunity to Shigella could be transferred only with lymphocytes; macrophages were found to be inactive.     

Tuberculosis DNA vaccine encoding Ag85A is immunogenic and protective when administered by intramuscular needle injection but not by epidermal gene gun bombardment

Immunogenicity and protective efficacy of a DNA vaccine encoding Ag85A from Mycobacterium tuberculosis were compared in BALB/c and C57BL (B6 and B10) mice immunized by intramuscular (i.m.) needle injection or epidermal gene gun (gg) bombardment. In BALB/c mice, gg immunization could induce elevated antibody and cytotoxic T lymphocyte responses with plasmid doses 50-fold lower than those required for i.m. immunization. Interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion, however, was much lower in gg-immunized than in i.m.-immunized BALB/c mice. On the other hand, C57BL mice reacted only very weakly to gg immunization, whereas elevated Ag85A-specific antibody, IL-2, and IFN-gamma responses (significantly higher than in BALB/c mice) were detected following vaccination by the i.m. route. Antibody isotypes were indicative of Th2 activation following gg injection of BALB/c and of Th1 activation following i.m. injection of C57BL mice. Finally, C57BL but not BALB/c mice were protected by i.m. Ag85A DNA immunization against intravenous M. tuberculosis challenge, as measured by reduced numbers of CFU in spleen and lungs, compared to animals vaccinated with control DNA. Gene gun immunization was not effective in either BALB/c or C57BL mice. These results indicate that i.m. DNA vaccination is the method of choice for the induction of protective Th1 type immune responses with the Ag85A tuberculosis DNA vaccine.     

Clinical and immunological study of percutaneous revaccination in children who originally received smallpox vaccine subcutaneously.

In a large multicenter smallpox vaccination study carried out from 1970 to 1973, it was found that 39% of children who were initially immunized by the subcutaneous route and then challenged percutaneously with a standard vaccination did not have measurable neutralizing antibody upon follow-up. Because of this finding, a percutaneous revaccination study was conducted at the St. Louis study center in 1975 and 1976. There were four study groups, which were composed on the basis of route of primary immunization (subcutaneous or percutaneous) and whether neutralizing antibody was detectable following the original percutaneous challenge immunization. Of 52 children revaccinated, all but four had accelerated reactions. There was no difference in size of lesions or day of maximum erythema among the four study groups. Only 66% of children originally vaccinated subcutaneously who did not have postchallenge neutralizing antibody had measurable neutralizing antibody following revaccination. Transformation studies with vaccinia viral antigen before and after revaccination were performed on lymphocytes from 50 children. There was no appreciable differences in responses either before or after revaccination when the four groups were compared. However, the mean stimulation ratio for the total group increased from 2.4 before revaccination to 4.6 3 weeks later. In primary subcutaneous vaccine recipients without pre-revaccination neutralizing antibody, lymphocyte transformation correlated directly with the neutralizing antibody response.