Immunoproteomic analysis of the murine antibody response to successful and failed immunization with live anti-Francisella vaccines

Francisella tularensis subspecies tularensis is one of the most virulent of bacterial pathogens for humans. Protective immunity against the pathogen can be induced in humans and some, but not all, mouse strains by vaccination with live, but not killed, vaccines. In mice, this protection is mediated predominantly by CD4+ and CD8+ T cells. This is thought to be the case too for humans. Nevertheless, it is possible that successful vaccination elicits antigen-specific antibodies that can serve as correlates of protection. To test this hypothesis we examined the repertoire of antibodies induced following successful immunization of BALB/c and CH3/HeN mice versus unsuccessful vaccination of C57BL/6 and DBA\2 mice with F. tularensis Live Vaccine Strain or following unsuccessful vaccination of BALB/c mice with highly related subspecies, F. novicida. The results showed that successful vaccination elicited antibodies to at least six proteins that were not recognized by antisera from vaccinated but unprotected mice.     

Sortase A Induces Th17-Mediated and Antibody-Independent Immunity to Heterologous Serotypes of Group A Streptococci

Group A streptococci (GAS) are associated with a variety of mucosal and invasive human infections. Recurrent infections by highly heterologous serotypes indicate that cross-serotype immunity is critical for prevention of GAS infections; however, mechanisms underlying serotype-independent protection are poorly understood. Here we report that intranasal vaccination of mice with Sortase A (SrtA), a conserved cell wall bound protein, reduced colonization of nasal-associated lymphoid tissue (NALT) by heterologous serotypes of GAS. Vaccination significantly increased CD4⁺ IL-17A⁺ cells in NALT and depletion of IL-17A by neutralizing antibody prevented GAS clearance from NALT which was dependent on immunization with SrtA. Vaccination also induced high levels of SrtA-specific antibodies; however, immunized, B cell-deficient mice cleared streptococcal challenges as efficiently as wild type mice, indicating that the cross-serotype protection is Th17-biased and antibody-independent. Furthermore, efficient GAS clearance from NALT was associated with a rapid neutrophil influx into NALT of immunized mice. These results suggest that serotype independent immune protection against GAS mucosal infection can be achieved by intranasal vaccination with SrtA and enhanced neutrophil function is critical for anti-GAS defense and might be a target for prevention of GAS infections.     

Trypanosoma cruzi: infection patterns in intact and athymic mice of susceptible and resistant genotypes

Inbred strains of mice inoculated with the T cruzi Y strain behaved as susceptible (A/J, C3H/HeN), intermediate (BALB/c) or relatively resistant (C57BL/6) with respect to the magnitude of parasitaemia and mortality rate. C57BL/10 mice were susceptible in relation to parasitaemia but resistant when mortality was analyzed. Infection with T cruzi CL strain presented the same results, except for C57BL/6 which behaved as susceptible mice. Athymic mice of various backgrounds revealed no differences in susceptibility, presenting the same dramatic parasitaemia, tissue colonization pattern and no inflammatory reaction in any of the tissues studied. Infection of euthymic and athymic BALB/c mice elicited the production of parasite-specific antibodies, which reached similar levels on the first 9 days but differed after day 13. Serum transfer experiments in BALB/c mice did not show great differences in parasitaemia but altered T. cruzi polymorphism reducing the slender forms in athymic mice. Histopathology of athymic BALB/c mice showed the same tissue tropism when infected either with T cruzi Y or CL strain.     

Susceptibility of several strains of mice to Echinostoma hortense infection

Susceptibilities of 5 different mice strains, including C3H/HeN, BALB/c, C57BL6, FvB and ICR, to Echinostoma hortense infection, was evaluated. The worm expulsion rate, worm size and egg production were observed from 1 to 8 weeks after infection with 30 metacercariae. C3H/HeN and ICR mice showed the highest worm maturation rates. The worm recovery rate and the number of eggs per gram (EPG) of feces was also higher in C3H/HeN and ICR mice than in BALB/c, C57BL6, and FvB mice. It is suggested that E. hortense is highly infectious to ICR and C3H/HeN mice, but not to the other strains of mice. Based on the results obtained, we believe that the susceptibility of different mouse strains to E. hortense infection is dependent on the genetic and immunologic background of mice.     

Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.     

Cutaneous leishmaniasis in anti-IgM-treated mice: enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway

The contribution of B cells and antibodies to either the resistance or susceptibility to cutaneous leishmaniasis has been investigated in mouse strains rendered B cell-deficient by treatment with anti-mouse IgM antisera from birth (mu-suppressed). These studies confirm that immunity to cutaneous disease in a normally resistant mouse strain (C3H/HeJ) is independent of antibody, but that B cells and/or antibodies are required for the evolution of suppressed DTH and the consequent disease susceptibility of BALB/c mice. Anti-IgM-treated BALB/c mice, which lacked detectable anti-leishmanial antibodies during the course of infection, displayed a sustained DTH response to leishmanial antigen and were able to control their cutaneous lesions. The enhanced resistance of mu-suppressed mice could be completely abrogated by transfer of suppressor T cells from infected control animals into mu-suppressed mice before their infection. Thus the suppressor T cells, which are generated during leishmanial infection in BALB/c mice, can effect suppression in the absence of antibody. Evidence that B cells or antibodies are required for the generation of suppressor T cells was demonstrated by using BALB/c mice in which suppressor T cells fail to be generated during infection as a result of prior sublethal irradiation. Splenic T cells from normal mice could overcome the resistance conferred by sublethal irradiation, whereas splenic T cells from mu-suppressed mice could not. Thus the enhanced resistance of mu-suppressed BALB/c mice appears to be a consequence of their lack of functional expression of a B cell-dependent T cell critical to the suppressor pathway.     

Sequences within the gag gene of mouse mammary tumor virus needed for mammary gland cell transformation

Previously, we identified a group of replication-competent exogenous mouse mammary tumor viruses that failed to induce mammary tumors in susceptible mice. Sequence comparison of tumorigenic and tumor-attenuated virus variants has linked the ability of virus to cause high-frequency mammary tumors to the gag gene. To determine the specific sequences within the gag gene that contribute to tumor induction, we constructed five distinct chimeric viruses that have various amino acid coding sequences of gag derived from a tumor-attenuated virus replaced by those of highly tumorigenic virus and tested these viruses for tumorigenic capacities in virus-susceptible C3H/HeN mice. Comparing the tumorigenic potentials of these viruses has allowed us to map the region responsible for tumorigenesis to a 253-amino-acid region within the CA and NC regions of the Gag protein. Unlike C3H/HeN mice, BALB/cJ mice develop tumors when infected with all viral variants, irrespective of the gag gene sequences. Using genetic crosses between BALB/cJ and C3H/HeN mice, we were able to determine that the mechanism that confers susceptibility to Gag-independent mammary tumors in BALB/cJ mice is inherited as a dominant trait and is controlled by a single gene, called mammary tumor susceptibility (mts), that maps to chromosome 14.     

Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major

C3H/HeN mice that are naturally resistant to cutaneous disease and systemic infections with the protozoan parasite, Leishmania major, were treated at the time of infection, and weekly thereafter, with mouse anti-rat IFN-gamma mAb or an irrelevant antibody of similar isotype. Anti-IFN-gamma-treated mice developed cutaneous lesions; parasites spread to the regional lymph nodes and then metastasized to spleens and livers. The course of disease in these animals was similar to that of genetically susceptible BALB/c mice. Two exceptions in the pathology of L. major infections were noted between BALB/c and anti-IFN-gamma-treated C3H/HeN mice: 1) BALB/c mice died of systemic complications, whereas C3H/HeN mice did not, and 2) multinucleated giant cells were observed in lymph nodes and spleens of infected BALB/c mice, whereas these cells were not observed in infected C3H/HeN mice. Control mice, those treated with either saline or irrelevant antibody of the same isotype as the anti-IFN-gamma monoclonal, showed no evidence of cutaneous disease (development of footpad lesions) or systemic infection (by histopathology). Total abrogation of the natural resistance of C3H/HeN mice could be achieved by treatment with as little as 0.5 mg/mouse/wk of anti-IFN-gamma antibody, or by a single dose of 1 mg/mouse anti-IFN-gamma antibody administered at the time of parasite inoculation. If antibody treatment was delayed for as little as 1 wk after parasite inoculation, the infections in treated animals resembled that of untreated or control antibody-treated mice: no cutaneous lesions (by footpad swelling or viable counts of leishmania in footpad tissue) or systemic disease (by microscopic analysis of touch preparations of internal organs, and histopathology of same). The production of IFN-gamma during the initial interaction of the parasite and host cells appears to be a major component of genetic control of natural resistance to infection with L. major in C3H/HeN mice.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.     

A novel block to mouse mammary tumor virus infection of lymphocytes in B10.BR mice

Classic studies on C57BL-derived mouse strains showed that they were resistant to mouse mammary tumor virus (MMTV) infection. Although one form of resistance mapped to the major histocompatibility complex (MHC) locus, at least one other, unknown gene was implicated in this resistance. We show here that B10.BR mice, which are derived from C57BL mice but have the same MHC locus (H-2^k) as susceptible C3H/HeN mice, are resistant to MMTV, and show a lack of virus spread in their lymphoid compartments but not their mammary epithelial cells. Although in vivo virus superantigen (Sag)-mediated activation of T cells was similar in C3H/HeN and B10.BR mice, T cell-dependent B-cell and dendritic cell activation was diminished in the latter. Ex vivo, B10.BR T cells showed a diminished capacity to proliferate in response to the MMTV Sag. The genetic segregation of the resistance phenotype indicated that it maps to a single allele. These data highlight the role of Sag-dependent T-cell responses in MMTV infection and point to a novel mechanism for the resistance of mice to retroviral infection that could lead to a better understanding of the interplay between hosts and pathogens.     

B lymphocytes are required for the generation of T cells that mediate healing of cutaneous leishmaniasis

The role that B lymphocytes and/or antibodies play in the healing of Leishmania major infections in genetically resistant C3H/HeN mice was investigated by monitoring the course of infection in animals that had been B cell depleted by treatment from birth with anti-IgM sera (mu-suppressed). L. major infection of mu-suppressed C3H/HeN mice produced lesions that were significantly larger than those induced in control animals, and failed to heal. Moreover, vaccinated mu-suppressed mice also developed chronic nonhealing infections, although their lesions were initially smaller than those developed by nonvaccinated mu-suppressed controls. The enhanced susceptibility of mu-suppressed mice could be completely overcome by adoptive transfer of T lymphocytes from mice that had spontaneously healed their lesions, and to a lesser extent by T lymphocytes from normal animals. Anti-leishmanial antibody responses were completely absent in mu-suppressed mice, regardless of whether they were lymphocyte reconstituted, whereas delayed type hypersensitivity (DTH) to leishmanial antigens was present in normal and mu-suppressed animals. The ability of immune T cells to protect mu-suppressed mice without restoring humoral responsiveness clearly indicates that antibodies are not necessary for healing leishmanial infections. Instead, the observed effect of mu-suppression argues that B lymphocytes are required for the generation of an effector T cell population, apparently unrelated to DTH, which mediates the healing of cutaneous lesions. These results thus provide the first evidence for the B cell and/or Ig dependency of a T cell population that is critical for the development of immunity against a microbial agent.     

Studies on immune responses to larval cestodes in mice. Increased susceptibility of certain mouse strains and hypothymic mice to Taenia taeniaeformis and analysis of passive transfer of resistance with serum.

Various inbred strains of mice vary markedly in their susceptibility to the larvae of the cestode, Taenia taeniaeformis. Males are generally more susceptible than females and the most susceptible common inbred mouse strains are those which are deficient in C5 and/or C4 components of complement. However, no genetic evidence is yet available to implicate loci controlling complement levels in susceptibility/resistance, and multiple genetic factors appear to be operative. Hypothymic, nu/nu ("nude") mice of the relatively resistant mouse strain, BALB/c, are highly susceptible in that cystic larvae in the liver develop in large numbers and more rapidly than in intact BALB/c.nu/+litter-mates. Cyclophosphamide pretreatment also increases the susceptibility of relatively resistant strains of mice in terms of both the number and size of liver cysts. Hypothymic and intact mice can be protected, absolutely, by an injection of serum from infected intact mice, provided the serum is given to recipient mice close to the time of oral egg administration. The protective activity of immune serum is absorbed totally by staphylococcal protein A-Sepharose columns and can be abolished by treatment of recipients with cobra venom factor. Cyst fluid from established larvae facilitates the activity of subhaemolytic amounts of guinea pig complement in a standard direct PFC assay. The data suggest that complement-fixing antibodies are responsible for inhibition of establishing larvae in mice and that one method of protection for established cystic larvae involves the alteration of host complement activity within the cyst.     

Evidence for separate genetic defects in C3H/HeJ and C3HeB/FeJ mice, that affect susceptibility to gram-negative infections

Past studies have suggested a linkage between susceptibility to Salmonella typhimurium infection and the Lpsd genotype in C3H mice. Recently, this linkage was questioned by the finding that C3HeB/FeJ mice (Lpsn,Lpsn) were highly susceptible to systemic S. typhimurium infection. The present study shows a marked difference between C3H/HeJ and C3HeB/FeJ in their susceptibility to Gram-negative urinary tract infection. The number of E. coli and S. typhimurium recovered from the kidneys 24 hr after infection was 70 to 100 times higher in C3H/HeJ than in C3HeB/FeJ or C3H/HeN mice. Subsequently, in C3HeB/FeJ mice S. typhimurium multiplied to the level of C3H/HeJ mice, resulting in a shorter mean survival time of C3H/HeJ and C3HeB/FeJ compared with C3H/HeN mice. In contrast, E. coli remained localized to the urinary tract of C3H/HeJ mice but were eliminated from C3HeB/FeJ and C3H/HeN mice. Thus, experimental E. coli urinary tract infection appears to provide a method to differentiate the genetic defects of C3H/HeJ and C3HeB/FeJ mice. The results support an influence of the Lpsd genotype on clearance of Gram-negative bacteria from the kidneys of C3H mice.     

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.     

TGF-beta-associated enhanced susceptibility to leishmaniasis following intramuscular vaccination of mice with Leishmania amazonensis antigens

Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.     

Host defense in cryptococcosis. II. Cryptococcosis in the nude mouse.

In the homozygous state, mice carrying the “nude” (nu) gene are hairless (nude), lack a thymus and have profound deficiency of cell-mediated immunity. Cryptococcosis was studied in BALB/c and Swiss mice, each strain carrying the nu gene. The purpose was to determine the interactions of the nu gene and mouse strain in terms of susceptibility to Cryptococcosis. Mice of both strains could be sensitized to produce delayed-type hypersensitivity reactions to cryptococcal extract in the heterozygous nu/X state, but not in the nu/nu state. Nu/X Swiss mice were more resistant than nu/X BALB/c mice to infection with a highly virulent strain (B) of Cryptococcus neoformans. However, nu/nu BALB/c and nu/nu Swiss mice were both highly susceptible to the same microorganism. Challenge with another cryptococcal strain (A) of much lower virulence for nu/X mice killed 100% of BALB/c and Swiss nu/nu mice. These studies indicate that thymus-dependent immune functions are critical determinants of host resistance to murine Cryptococcosis.     

Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine

Stockdale P. G. H., Stockdale M. J., Rickard M. D. and Mitchell G. F. 1985. Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine, International Journal for Parasitology15: 447–452. Five inbred strains of mice and three hypothymic (nude) strains were infected orally with different doses of E. falciförmis oocysts. After resolution of primary infection as determined by faecal oocyst output, mice were challenged orally with a second dose of E. falciformis. Amongst the intact mice, BALB/c proved the most resistant to primary infection, while C3H/He mice were most susceptible, in terms of faecal oocyst production. Resistance was far more dramatic in BALB/c mice given high numbers of challenge oocysts. In terms of mortality at high oocyst doses, CBA/H were the most susceptible. All of the strains of mice were highly resistant to reinfection. In the case of nude mice, BALB/c. nu/nu were more susceptible than CBA/H.nu/nu or C57BL/6.nu/nu both in terms of faecal oocyst production and mortality. Thus the most resistant inbred mouse strain (BALB/c) is the least resistant in the absence of T cells. Unlike intact mice, nude mice showed no resistance to reinfection, this result being in line with previous work on this and other Eimeria spp. in nude mice.     

Age Dependence of Immunity Induced by a Candidate Universal Influenza Vaccine in Mice

Influenza has a major impact on the elderly due to increased susceptibility to infection with age and poor response to current vaccines. We have studied universal influenza vaccine candidates based on influenza A nucleoprotein and matrix 2 (A/NP+M2). Long-lasting protection against influenza virus strains of divergent subtypes is induced, especially with mucosal immunization. Here, we tested universal vaccination in BALB/c mice of different ages. Vaccination used intramuscular DNA priming to A/NP+M2 followed by intranasal (i.n.) boosting with recombinant adenoviruses (rAd) expressing the same antigens, or only A/NP+M2-rAd given i.n. Antigen-specific systemic antibody responses were induced in young, middle-aged, and elderly mice (2, 11–17, and 20 months old, respectively), but decreased with age. Antibody responses in bronchoalveolar lavage (BAL) were detected only in young mice. Antigen-specific T cell responses were seen in young and middle-aged but not elderly mice. A/NP+M2 vaccination by the two regimens above protected against stringent challenge in young and middle-aged mice, but not in elderly mice. However, mice vaccinated with A/NP-rAd or A/M2-rAd during their youth were partially protected against challenge 16 months later when they were elderly. In addition, a regimen of two doses of A/NP+M2-rAd given i.n. one month apart beginning in old age protected elderly mice against stringent challenge. This study highlights the potential benefit of cross-protective vaccines through middle age, and suggests that their performance might be enhanced in elderly individuals who had been exposed to influenza antigens early in life, as most humans have been, or by a two-dose rAd regimen given later in life.