Enhancing activity of mycobacterial cell-derived adjuvants on immunogenicity of recombinant human hepatitis B virus vaccine

In a previous study, we demonstrated that a lipophilic derivative of muramyl dipeptide [MDP-Lys(L18)] augmented antibody response to recombinant human hepatitis B surface antigen (rhHBsAg) when it was co-immunized with rhHBsAg solubilized in PBS. Here, we examined adjuvant activity of two bacterial cell-derived adjuvants such as Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) and trehalose-6,6'-dimycolate (TDM), to enhance immunogenicity of rhHBsAg, comparing their activity with that of MDP-Lys(L18). In an animal model where mice were immunized subcutaneously (s.c.) with rhHBsAg (25 micrograms/mouse) admixed with 100 micrograms/mouse of BCG-CWS (Vac/BCG-CWS) or 50 micrograms/mouse of TDM (Vac/TDM) in o/w emulsion formulation, both mice immunized with Vac/BCG-CWS and Vac/TDM showed higher antibody titres to HB antigen than those of mice immunized with the recombinant vaccine alone. The activity of BCG-CWS and TDM to enhance antibody induction seemed to be almost the same with that of MDP-Lys(L18). Furthermore, the enhanced antibody response raised by these adjuvants was shown to be due to high titres of HB antigen-specific IgG1. In addition, the activity of these three adjuvants to enhance antibody response was shown to be higher than that of the present clinical vaccine, aluminium hydroxide-attached rhHBsAg (rhHBsAg-alum). In an analysis of delayed-type hypersensitivity (DTH) reaction where mice were immunized with rhHBsAg admixed with or without each adjuvant in o/w emulsion and followed by intrafootpad (i.f.) injection of rhHBsAg 4 weeks after immunization, mice immunized with Vac/BCG-CWS and Vac/TDM as well as Vac/MDP-Lys(L18) showed a significant increment of swelling reaction. These results suggest that BCG-CWS, TDM and MDP-Lys(L18) are potential adjuvants to enhance the immunogenicity of rhHBsAg to induce humoral and cellular responses.     

The muramyl dipeptide analog GMTP-N-DPG preferentially induces cellular immunity to soluble antigens

GMTP-N-DPG (N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamyl- L-alanyl-dipalmitoylpropylamide) is a lipophilic derivative of the immunologically active compound MDP and has adjuvant properties. GMTP-N-DPG was compared with other adjuvants in model vaccine systems using ovalbumin (OVA) and a synthetic peptide derived from pp89 of murine cytomegalovirus as antigens. When serum from C57/Bl mice immunized with OVA was tested for the presence of anti-OVA antibody, samples from mice immunized with OVA plus GMTP-N-DPG had ELISA optical density (O.D.) readings twice as high as those from mice immunized with antigen alone. In contrast, samples from mice immunized with the liposomal monophosphoryl lipid A (MPL) formulation exhibited ELISA O.D. readings tenfold higher than samples from mice immunized with antigen alone. Relative levels of specific antibody in serum samples from mice immunized with OVA plus the saponin adjuvant QS-21 were equal to the GMTP-N-DPG samples. When spleen cells from immunized mice were tested for their proliferative response to OVA, we found that liposomal MPL was again the optimal adjuvant, whereas the proliferative responses of cells from mice immunized with GMTP-N-DPG or QS-21 were no better than cells from mice immunized with OVA alone. In contrast to the relatively low antibody and proliferation levels, spleen cells from mice immunized with GMTP-N-DPG and OVA demonstrated the highest level of anti-OVA CTL activity. Spleen cells from mice immunized with the pp89 peptide plus GMTP-N-DPG also exhibited CTL activity. Using antibody and complement mediated cytotoxicity it was determined that the CTL were CD8+. Based on these results, we believe that GMTP-N-DPG may be an excellent candidate adjuvant in vaccines for diseases in which a strong cell-mediated response is desired.     

Antibody response to pneumococcal polysaccharide vaccine in young, adult and old mice

The anti-pneumococcal antibody response was studied in young (5-week-old) and adult (10-week-old) BALB/c and CBA/J mice and in adult (9-10-week-old) and old (12-, 18- and 24-month-old) AB6F1 and B6D2F1 mice after s.c. immunization with a 23-valent pneumococcal polysaccharide vaccine. Both young and adult mice showed a significant IgM antibody response to the vaccine 6 days after immunization with 1-11 micrograms antigen. There were significant immune responses to serotypes 1, 2, 4 and 7F in contrast to small responses to serotypes 14, 19F and 23F after immunization with the vaccine. One month after immunization, there were only marginal differences in IgM anti-pneumococcal antibody levels to the vaccine (anti-PPS) between immunized and unimmunized BALB/c mice, whereas in CBA/J mice the anti-PPS remained higher in immunized than in unimmunized mice. Immunization of old mice induced a significant IgM antibody response 6 days after immunization, but the anti-PPS thereafter decreased rapidly towards preimmunization values in AB6F1 mice. A significant IgG anti-PPS was not detected in any of the mice studied. The IgA anti-PPS tended to vary over time with no consistent pattern. It is important to carefully consider age and strain of the mice used when studying the immune response to pneumococcal polysaccharide antigens.     

Nasal immunization with group B streptococci can induce high levels of specific IgA antibodies in cervicovaginal secretions of mice

We have studied the cervicovaginal antibody responses in mice, by ELISA, following mucosal immunizations with group B streptococci (GBS) serotype III/R4. Immunizations were carried out either: (1) rectally with GBS alone; (2) rectally with GBS plus cholera toxin (CT); (3) nasally with GBS alone; (4) nasally with GBS+CT; or (5) nasally under general anesthesia with GBS+CT. Nasal immunizations with GBS alone led to at least tenfold higher levels of specific IgA-antibodies to GBS in cervicovaginal secretions than with any other immunization. These mucosal antibody levels were higher than after rectal immunizations, and 2-17 times higher than the corresponding IgA antibody levels in sera. Markedly lower cervicovaginal antibody levels were found in mice which had received GBS together with CT as a mucosal adjuvant than in mice immunized by the same routes with GBS alone. Our observations indicate that a nasal vaccine consisting of GBS might induce sufficient antibody levels to protect against genital colonization of these bacteria.     

A candidate Ross River virus vaccine: preclinical evaluation

A killed Ross River virus vaccine is being developed in an effort to prevent the, ca 5000 cases of epidemic polyarthritis which occur in Australia each year. The symptoms of epidemic polyarthritis commonly last 30-40 weeks and 25% of patients have symptoms for a year or more. There is no cure. Although there was some strain to strain variation, particularly after a single injection, outbred and inbred strains of mice all produced significant levels of anti-Ross River virus antibody after intramuscular (i.m.) injection with 24 h BEI inactivated, sucrose gradient purified, Ross River virus vaccine. Mice immunized i.m. with two 20 micrograms doses of vaccine or live virus produced similar levels of neutralizing antibody but the reaction of IgG 2a and IgG 2b antibody from these two groups of mice to Ross River virus proteins in western blots differed. Antibody from BALB/c mice immunized with this vaccine neutralized all strains of Ross River virus tested, in vitro, albeit to different degrees.     

Cross-clade p17 peptide recognition by antisera to HGP-30, a 30-amino acid synthetic peptide antigen from HIV type 1 p17

HGP-30, a 30-amino acid synthetic peptide analog of HIV-1SF2 p17 (aa 86-115), was used to immunize both mice and humans. Since the amino acid sequence of HGP-30 is relatively conserved among different HIV-1 strains and clades, experiments were carried out to determine if antisera obtained by immunizing animals and humans can recognize HGP-30-related peptide consensus sequences belonging to different clades. Results show that antisera from mice immunized with HGP-30 can recognize clade B and C and to a lesser degree clade A and E consensus sequences of HIV-1, in addition to recognizing HGP-30 sequence. The cross-clade recognition was higher in mouse sera obtained on day 42 than on day 14 or 28. MPL/SE and Novasomes were better adjuvants than alum in inducing antibodies that showed cross-clade recognition and IgG2a and IgG2b antibody isotypes. Similar cross-clade recognition was observed in several sera from humans immunized with an HGP-30/KLH/alum formulation. The human sera from HGP-30-immunized subjects evaluated for cross-clade recognition of HGP-30 peptides were from subjects whose cells showed significant protection from HIV infection on virus challenge in the hu-PBL-SCID mouse model. These studies suggest that HGP-30 may be useful as a candidate vaccine antigen for populations in countries with prevalence of different HIV clades.     

Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 microg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 microg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 microg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 microg. The CTLs induced in BALB/c mice immunized twice with 100 microg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

gamma/delta T cell-deficient mice have impaired mucosal immunoglobulin A responses

Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCRdelta-/-) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCRdelta-/- mice. The TCRdelta-/- mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toxoid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toxoid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody-producing cells were present in the intestinal lamina propria and Peyer's patches of TCRdelta-/- mice compared with the orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta cells in mucosal immunity.     

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1-14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0-10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.     

Human immune responses to influenza virus vaccines administered by systemic or mucosal routes

Healthy adult volunteers were immunized by parenteral or oral routes with trivalent inactivated influenza vaccine (A/Chile/1/83 (H1N1), A/Mississippi/1/85 (H3N2), and B/Ann Arbor/1/86), or intranasally with live attenuated, cold-adapted influenza type A/Texas/1/85 (H1N1) reassortant virus. In all volunteers, cells spontaneously secreting IgA, IgG or IgM antibodies specific to influenza virus were detected in peripheral blood on days 6-13 after immunization, and specific IgA, IgG and IgM antibodies to influenza vaccine were measured in sera and external secretions (saliva and nasal lavage). Following systemic immunization, a raise in specific antibodies of all isotypes was observed in sera beginning on day 13. Although small variations in IgA and IgM antibodies in saliva and nasal lavages were detected, antigen-specific IgG significantly increased between days 13 and 27. Intranasal administration of attenuated virus induced IgA and IgG antibodies in serum as well as in secretions. Serum antibodies were not substantially influenced by oral immunization, only a small increase in all isotypes was observed in volunteers' sera 21 days after ingestion of vaccine. However, in secretions, antigen-specific IgA and IgG responses were detected one week after immunization and reached a peak response on day 20. These studies show that different routes of immunization can be effective for the induction of specific antibodies, and support the concept of the common mucosal immune system in humans by demonstrating that the oral or intranasal administration of antigen-induced specific antibodies of IgA isotype in external secretions, preceded by the transient appearance in peripheral blood of specific antibody-producing cells.     

Kinetics of hepatitis B surface antigen-specific immune responses in acute and chronic hepatitis B or after HBs vaccination: stimulation of the in vitro antibody response by interferon gamma

Because cellular and humoral immune responses against the hepatitis B virus (HBV) surface antigen (HBs) might be crucial to overcome HBV infection, HBs-specific B- and T-cell responses of HBV patients and HBs vaccine recipients were analyzed quantitatively and functionally. In patients with acute hepatitis B (AHB), transient high anti-HBs-secreting B-cell frequencies were observed early after clinical onset, whereas 1 patient who probably developed chronic infection and chronic HBV carriers had absent or weak B- and T-cell responses. In HBs vaccine recipients, maximal HBs-specific B- and T-cell responses were detected after the first injection that decreased gradually before anti-HBs antibodies appeared in serum. Years after vaccination, anti-HBs-secreting B cells were enriched in the bone marrow. After in vitro stimulation with HBsAg, peripheral blood mononuclear cells (PBMC) of only 1 of 5 acute and 1 of 6 chronic HBV patients, but of all 6 vaccine recipients, secreted varying amounts of interferon gamma (IFN-gamma), but no interleukin-4 (IL-4) or IL-5. Furthermore, the addition of IFN-gamma, but not of IL-2, -4, -12, or IFN-alpha, resulted in strong increases of anti-HBs-secreting B cells in vaccine recipients and chronic carriers. In conclusion, circulating anti-HBs-secreting B cells were significantly higher in early acute hepatitis B or early after HBs vaccination than in chronic hepatitis B and decreased in the follow-up as a result of compartmentalization to lymphoid tissues. Release of IFN-gamma by antigen-stimulated T cells might be critical for anti-HBs formation.     

Human diploid cell culture rabies vaccine (HDCV) and purified chick embryo cell culture rabies vaccine (PCECV) both confer protective immunity against infection with the silver-haired bat rabies virus strain (SHBRV)

The demonstration of extensive differences in the antigenic makeups of the silver-haired bat rabies virus (SHBRV) and canine rabies virus (COSRV) strains raised concerns as to whether current licensed rabies vaccines are sufficiently protective against SHBRV. NIH mouse protection test results show that both the human diploid cell culture rabies vaccine (HDCV) and the purified chicken embryo cell rabies vaccine (PCECV) protected against lethal infection with SHBRV as well as the canine rabies strain COSRV. However, in this investigation, the potencies of both vaccines in mice were found to be significantly higher for COSRV than for SHBRV. The in vivo protection data are confirmed by in vitro virus neutralizing antibody (VNA) test results which demonstrate that mice immunized with HDCV or PCECV develop significantly higher VNA titres against COSRV than against SHBRV. In contrast, VNA tests of sera from individuals immunized with HDCV or PCECV showed that humans, as opposed to mice, develop significantly higher VNA titres against SHBRV than against COSRV. These data suggest that HDCV and PCECV will protect humans against infection with the silver-haired but rabies virus strain in addition to canine rabies virus strains.     

CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.     

Evaluation of live, cold-adapted influenza A and B virus vaccines in elderly and high-risk subjects

We have evaluated the use of live cold-adapted influenza A and B virus vaccines in the elderly. Cold-adapted influenza A and B virus vaccines are safe and modestly immunogenic in individuals over 65 years of age. However, our studies and those of other groups have shown that immune response to cold-adapted vaccines in this age group are modest. Administration of combined cold-adapted influenza A and inactivated influenza vaccine has resulted in slightly higher frequencies of local and systemic humoral immune responses than inactivated vaccine alone in some, but not all, studies. In a double-blind field trial conducted in nursing homes over a 3 year period, combined cold-adapted influenza A (H3N2) and trivalent inactivated influenza vaccine resulted in a 60% decrease (95% CI, 18-82%) in the rate of laboratory documented influenza A compared with inactivated vaccine alone. Further studies of multivalent cold-adapted influenza vaccines used in combination with inactivated vaccine should be performed.     

Influenza virus neutralizing antibodies and IgG isotype profiles after immunization of mice with influenza A subunit vaccine using various adjuvants

The influence of various adjuvants on the development of influenza virus neutralizing antibodies and distribution of anti-influenza virus IgG isotypes after immunization of mice with influenza A (H3N2) subunit vaccine was investigated. Serum titres of influenza virus neutralizing antibodies and titres of influenza specific IgG isotypes were determined by a neutralization enzyme immunoassay (N-EIA) and a cell-associated antigen enzyme immunoassay (CA-EIA), respectively. Serum antibody titres as measured by the two tests correlated highly (r = 0.82; P < 0.001). N-EIA titres were enhanced by 38- and 34-fold, when L180.5/RaLPS and FCA, respectively, were administered with 1 microgram of vaccine. The adjuvants Q-VAC, L180.5 [W/O/W], L180.5 alone and Montanide ISA 740 were only moderately or not effective in enhancing the immune response to the 1 microgram dose of vaccine. The Q-VAC and L180.5/RaLPS adjuvants favoured IgG2a and IgG2b isotype responses to influenza compared to the other adjuvants. We suggest that N-EIA and CA-EIA may be valuable tools to monitor the effects of adjuvants on the neutralizing antibody and antibody isotype responses after influenza vaccination.     

Hepatitis B vaccine containing surface antigen and selected preS1 and preS2 sequences. 1. Safety and immunogenicity in young, healthy adults

The safety and immunogenicity of a yeast-derived recombinant hepatitis B virus (HBV) vaccine containing surface antigen (S) and selected preS1 and preS2 sequences (S-L*) were compared with those of a vaccine prepared with S alone (Engerix-B). S-L* consisted of composite particles containing S and L* at a ratio of 70/30. L* encompassed amino acid residues 12-52 of preS1 residues 133-145 of preS2, and the entire S domain. A total of 100 healthy, HBV-seronegative, young adults were randomized to receive 20 micrograms/dose of either S-L* or Engerix-B under double-blind conditions according to a 0-, 1-, 2-, 12-month schedule. In vivo humoral and in vitro lymphoproliferative responses to S and preS regions were monitored. Addition of the selected preS sequences to S did not enhance the in vivo humoral anti-HBs response but improved the in vitro stimulating capacity of the antigen (L*) in S-L* primed subjects.     

Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms

Live-attenuated retroviruses have been shown to be effective retroviral vaccines, but currently little is known regarding the mechanisms of protection. In the present studies, we used Friend virus as a model to analyze characteristics of a live-attenuated vaccine in protection against virus-induced disease. Highly susceptible mice were immunized with nonpathogenic Friend murine leukemia helper virus (F-MuLV), which replicates poorly in adult mice. Further attenuation of the vaccine virus was achieved by crossing the Fv-1 genetic resistance barrier. The minimum dose of vaccine virus required to protect 100% of the mice against challenge with pathogenic Friend virus complex was determined to be 10(3) focus-forming units of attenuated virus. Live vaccine virus was necessary for induction of immunity, since inactivated F-MuLV did not induce protection. To determine whether immune cells mediated protection, spleen cells from vaccinated donor mice were adoptively transferred into syngeneic recipients. The results indicated that immune mechanisms rather than viral interference mediated protection.     

Simultaneous determination of pteridines in multicomponent mixtures using derivative spectrophotometry and partial least-squares calibration

Murine phosphatidyl choline (PtC)-specific B cells in normal mice belong exclusively to the B-1 subset. Analysis of anti-PtC (VH12 and VH12/Vkappa4) transgenic (Tg) mice indicates that exclusion from B-0 (also known as B-2) occurs after immunoglobulin gene rearrangement. This predicts that PtC-specific B-0 cells are generated, but subsequently eliminated by either apoptosis or differentiation to B-1. To investigate the mechanism of exclusion, PtC-specific B cell differentiation was examined in mice expressing the X-linked immunodeficiency (xid) mutation. xid mice lack functional Bruton's tyrosine kinase (Btk), a component of the B cell receptor signal transduction pathway, and are deficient in B-1 cell development. We find in C57BL/ 6.xid mice that VH12 pre-BII cell selection is normal and that PtC-specific B cells undergo modest clonal expansion. However, the majority of splenic PtC-specific B cells in anti-PtC Tg/xid mice are B-0, rather than B-1 as in their non-xid counterparts. These data indicate that PtC-specific B-0 cell generation precedes segregation as predicted, and that Btk function is required for efficient segregation to B-1. Since xid mice exhibit defective B cell differentiation, not programmed cell death, these data are most consistent with an inability of PtC-specific B-0 cells to convert to B-1 and a single B cell lineage.     

Interactions of itraconazole with amphotericin B in the treatment of murine invasive candidiasis

The interactions of amphotericin B and itraconazole were studied in murine invasive candidiasis. Candida albicans-infected mice were treated for 10 consecutive days, 24 h after infection. Survival was monitored over 30 days and kidney cultures were done. Mice treated with amphotericin B (0.2 mg/kg/day intraperitoneally) or itraconazole (100 mg/kg/day by oral gavage in two divided doses/ day) had a 30-day survival of 20% or 40%. Concomitant administration of both drugs resulted in 100% mortality; 90% of mice treated with amphotericin B (1 mg/kg/day) survived. With the combination, 100% were dead by day 28 (P < or = .001 vs. amphotericin B). With sequential therapy (i.e., 5 days with one drug and then 5 days with the other), survival was inferior to that with amphotericin B alone but similar to that with itraconazole alone. Kidney culture results confirmed the antagonism of the combination compared with amphotericin B alone. In treatment of murine invasive candidiasis, the concomitant or sequential use of amphotericin B and itraconazole results in a negative interaction.     

Routine amnioscopy at term

Coxsackievirus B3 infection in pregnant mice leads to a severe pancreatitis with a retardation of foetal growth and increased wastage. The present study demonstrates that animals may be immunized actively or passively against this infection to allow foetal development to proceed normally. Active immunization was achieved by injecting a low dose of live virus into 4-week-old animals. These mice were then mated at 10 weeks and given a high dose of virus on the eighth day of pregnancy. Examination at 18 days' gestation revealed that foetal growth was not significantly different from the controls injected with heat-killed virus, and pathological changes in the mothers were not seen. Animals were passively immunized against Coxsackievirus B3 in pregnancy by injecting serum from immunized animals 1 day before the high dose of live virus was given. This procedure also protected against the effects of the virus and litter sizes and foetal weights were normal.