Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial.

Orally delivered, inactivated whole-cell vaccines are safe methods of inducing local and systemic immunity. To increase surface proteins associated with adherence and invasion, Shigella sonnei were grown in BHI broth containing deoxycholate. A whole-cell vaccine (SsWC) was then produced by formalin inactivation. In pre-clinical studies, the SsWC vaccine was immunogenic and protected against S. sonnei-induced keratoconjunctivitis in the guinea pig model. In a randomized, double-blind, placebo-controlled, Phase I study, 10 evaluable subjects received either three doses of SsWC on Days 0, 14, and 28 (N = 3); five doses of SsWC on Days 0, 2, 4, 6, and 28 (N = 4); or placebo (N = 3). Each dose contained 2.0 x 10(10) inactivated cells. Serum and fecal antibodies against SsWC, LPS, and IpaC were measured by ELISA. A > or = 4-fold increase in titer was considered significant. Both SsWC dosing regimens were well tolerated. No fever or severe gastrointestinal symptoms were noted by any of the vaccinated subjects. Antibody responses were similar in the two dosing groups. Serum IgG or IgA responses to SsWC were seen in six of seven vaccinees (86%), to LPS in four of seven (57%), and to IpaC in five of seven (61%). Fecal IgA responses to these three antigens developed in five of five, three of five, and three of five subjects, respectively. Among the seven vaccinees, geometric mean rises in serum IgA levels to all three immunogens were significant; IgG increases trended toward significance (paired one-tailed t-test). We conclude that SsWC was immunogenic and protective in animal studies and well tolerated and immunogenic in a Phase I trial.

Pneumococcal surface protein A is expressed in vivo, and antibodies to PspA are effective for therapy in a murine model of pneumococcal sepsis.

Pneumococcal surface protein A (PspA) is an immunogenic protein expressed on the surface of all strains of Streptococcus pneumoniae (pneumococcus) and induces antibodies which protect against invasive infection in mice. Pneumococci used for infectious challenge in protection studies are typically collected from cultures grown in semisynthetic medium in vitro. The purpose of these studies is to confirm that PspA is expressed by pneumococci during growth in vivo at a level sufficient for antibodies to PspA to be protective. Mice were actively immunized with purified PspA or by passive transfer of monoclonal antibody (MAb) and challenged with a capsular type 3 strain in diluted whole blood from bacteremic mice. All were protected against challenge with 10 times the 50% lethal dose (LD(50)), and mice challenged with 1,000 times the LD(50) had increased survival compared with controls. Additionally, nonimmune mice treated with MAbs to PspA or PspA immune serum at 6 and 12 h after infection with 10 times the LD(50) also showed increased survival. Northern blot analysis of RNA from pneumococci grown either in vitro or in vivo showed similar levels of PspA mRNA. These results demonstrate that PspA is expressed in vivo in a mouse model and that immunization with PspA induces antibodies to an antigen which is expressed during the course of invasive infection. Immunotherapy with antibodies to PspA may have some utility in treating pneumococcal infections in humans.

Immunization of humans with recombinant pneumococcal surface protein A (rPspA) elicits antibodies that passively protect mice from fatal infection with Streptococcus pneumoniae bearing heterologous PspA.

Pneumococcal surface protein A (PspA), a cross-reactive protein expressed by all pneumococci, is known to elicit an antibody in animals that can passively protect mice from infection with Streptococcus pneumoniae. A phase I trial with recombinant PspA showed the protein to be immunogenic in humans. Pre- and postimmune serum samples from this trial were examined, and human antibody to PspA could protect mice from pneumococcal infection. The serum samples of subjects immunized twice with 125 microg of PspA had >100 times as much antibody per milliliter as was required to consistently protect mice from fatal infection (1.3 microg/dose). At least 98% of PspAs fall into PspA sequence/serologic families 1 or 2. Human antibodies elicited by a family 1 PspA protected against infection with S. pneumoniae expressing either family 1 or 2 PspAs and with strains of all 3 capsular types tested: 3, 6A, and 6B. These studies suggest that PspA may have efficacy as a human vaccine.

Immunization of healthy adults with a single recombinant pneumococcal surface protein A (PspA) variant stimulates broadly cross-reactive antibodies to heterologous PspA molecules.

Pneumococcal surface protein A (PspA) is a highly variable protein found on all strains of pneumococci. To be successful, a PspA-based vaccine for S. pneumoniae must induce antibodies that are broadly cross-reactive. To address whether cross-reactive antibodies could be induced in man, we evaluated serum from adults immunized with recombinant clade 2 PspA from strain Rx1. Immunization with 5-125 microg rPspA lead to a significant increase in circulating anti-PspA antibodies, as well as antibodies reactive to heterologous rPspA molecules. Increased binding of post-immune sera to 37 pneumococcal strains expressing a variety of PspA and capsule types was observed, versus pre-immune sera. The extent of cross-clade reactivity of human anti-rPspA followed roughly the amount of sequence homology to the non-clade 2 antigens. It is hypothesized that priming of humans by natural exposure to S. pneumoniae contributes to the breadth of the cross-reactivity of antibody to PspA.

The potential to use PspA and other pneumococcal proteins to elicit protection against pneumococcal infection.

Pneumococcal proteins, alone, in combination with each other, or in combination with capsular polysaccharide-protein conjugates may be useful pneumococcal vaccine components. Four proteins with a potential for use in vaccines are PspA, pneumolysin, PsaA, and PspC. In a mouse model of carriage, PsaA and PspC were the most efficacious vaccine proteins. Of these, PsaA was the best at eliciting protection against carriage. However, a combination of PspA and pneumolysin may elicit stronger immunity to pulmonary infection and possibly sepsis than either protein alone. Recently, a phase one trial of a recombinant family 1 PspA was completed in man. PspA was observed to be safe and immunogenic. Injection of 0.1 ml of immune serum diluted to 1/400 was able to protect mice from fatal infection with S. pneumoniae. Under these conditions, pre-immune serum was not protective. The immune human serum protected mice from infections with pneumococci expressing either of the major PspA families (1 and 2) and both of the pneumococcal capsular types tested: 3 and 6.

The potential for using protein vaccines to protect against otitis media caused by Streptococcus pneumoniae.

Potential vaccine strategies against otitis media are to prevent (1) symptomatic infections in the middle ear and/or (2) carriage of pneumococci and thereby subsequent middle ear infections. The possibility of using immunity to virulence proteins of pneumococci to elicit immunity against pneumococci has been examined. PspA has been found to have efficacy against otitis media in animals. Vaccination with a mixture of PsaA and PspA has been observed to offer better protection against nasal carriage in mice, than vaccination with either protein alone. PspA and pneumolysin have been shown to elicit protection against invasive infections. The inclusion of a few of these proteins into the polysaccharide-protein conjugate vaccines may be able to enhance their efficacy against otitis media and might be able to constitute a successful all-protein pneumococcal vaccine.

Modulating ongoing Th2-cell responses in experimental leishmaniasis.

Dramatically polarized T helper (Th)-cell responses are seen in experimental murine infections with Leishmania major. Resistant mice develop a Th1-cell type response and heal the primary lesion, while susceptible mice develop non-protective Th2-cell responses, and the disease eventually proves fatal. Deservedly, much effort has gone into determining factors that influence the development of these T-cell subsets early in infection; however, little is known about how the polarity of established responses can be permanently modified. In this article, Gary Nabors reviews his findings on modifying ongoing Th2-cell responses in susceptible mice, and discusses therapies that have proven effective involving reducing the level of infection using a conventional antileishmanial drug, combined with agents that push the response towards the Th1 pole.

Successful chemotherapy in experimental leishmaniasis is influenced by the polarity of the T cell response before treatment.

Little is known about the influence of host factors on successful chemotherapy in leishmaniasis. Although successfully treated patients will convert from a delayed-type hypersensitivity (DTH)-negative to a DTH-positive state, the importance of the immune status of the host before treatment remains largely unexplored. In experimental murine cutaneous leishmaniasis, it was found that increased polarization towards a Th2 cytokine profile before the onset of drug therapy leads to an increased frequency of relapse after treatment. Whereas >50% of mice with established Leishmania major infections were cured when treated with the antileishmanial drug sodium stibogluconate, <10% of mice were cured when the animals had been pretreated with anti-interferon-gamma antibody to polarize the response toward a Th2 cytokine pattern before therapy. With successful drug therapy, cytokine profiles were found to switch from a Th2 to Th1 pattern, and resistance to reinfection was observed.

The influence of the site of parasite inoculation on the development of Th1 and Th2 type immune responses in (BALB/c x C57BL/6) F1 mice infected with Leishmania major.

Although inbred strains of mice are classified as genetically resistant or susceptible to Leishmania major based upon their ability to control infection, other factors such as the strain, dose, and site of parasite inoculation can also affect the outcome of the disease. Here we used the F1 progeny of BALB/c (susceptible) and C57BL/6 (resistant) mice (designated CB6F1) to investigate whether mice or intermediate susceptibility to infection differed from the parental strains in their ability to control infections at different cutaneous sites. CB6F1 mice developed progressive disease when inoculated in the dorsal skin, but healed infections in the footpad. Consistent with these observations, mice inoculated in the footpad ultimately developed Th1 responses, known to be required for healing, while Th2 responses developed in mice inoculated in the dorsal skin. However, IL-4 and IFN-gamma production during the first few weeks of infection was similar in CB6F1 mice inoculated at either site, suggesting that factors in addition to the relative levels of these cytokines produced early in infection may influence the nature of the antileishmanial immune response, and the eventual disease outcome. Infection in CB6F1 mice provides a model for the study of immunity to L. major in genetically identical animals, in which a prolonged mixed Th1/Th2 cytokine pattern initially develops, but ultimately diverges into more defined Th1 and Th2 type responses.

Activity of pentostam (sodium stibogluconate) against cutaneous leishmaniasis in mice treated with neutralizing anti-interferon-gamma antibody.

Studies with Leishmania donovani in the mouse have demonstrated that an intact T cell compartment is required for effective anti-leishmanial therapy using pentavalent antimony compounds such as Pentostam (sodium stibogluconate), suggesting that the in vivo efficacy of drug treatment is at least partially immune-based. Similarly, Leishmania-infected, immunodeficient human patients including those with acquired immunodeficiency syndrome (AIDS) generally relapse following therapy with antimonials. However, sodium stibogluconate is directly parasiticidal in vitro, in the absence of T cells or T cell products. Using a model of a cutaneous form of leishmaniasis, in which susceptible BALB/c mice were infected with Leishmania major, we investigated whether the antileishmanial activity of the drug demonstrated a requirement for interferon-gamma (IFN-gamma), a cytokine produced during a T helper cell type 1 (Th1) immune response and known to contribute to resistance to infection, and whether drug therapy affected the nature of the antileishmanial response. Lesion development was suppressed in mice treated from the onset of infection with sodium stibogluconate alone, and in animals treated with sodium stibogluconate plus a neutralizing anti-IFN-gamma antibody, and tissue parasite burdens were approximately 10,000-fold less at the end of therapy in both groups compared with controls. Lesion development was similarly suppressed in mice with established lesions treated with either sodium stibogluconate alone, or sodium stibogluconate plus anti-IFN-gamma antibody. The production of IFN-gamma by cells from infected animals was somewhat increased immediately following therapy with sodium stibogluconate, an effect that was not long-lasting, while interleukin-4 (IL-4) production was not affected by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Switch from a type 2 to a type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy with interleukin 12 and Pentostam.

Successful treatment in allergic, autoimmune, and infectious diseases often requires altering the nature of a detrimental immune response mediated by a particular CD4+ T helper (Th) cell subset. While several factors contribute to the development of CD4+ Th1 and Th2 cells, the requirements for switching an established response are not understood. Here we use infection with Leishmania major as a model to investigate those requirements. We report that treatment with interleukin 12 (IL-12), in combination with the antimony-based leishmanicidal drug Pentostam, induces healing in L. major-infected mice and that healing is associated with a switch from a Th2 to a Th1 response. The data suggest that decreasing antigen levels may be required for IL-12 to inhibit a Th2 response and enhance a Th1 response. These observations are important for treatment of nonhealing forms of human leishmaniasis and also demonstrate that in a chronic infectious disease an inappropriate Th2 response can be switched to an effective Th1 response.

Depletion of interleukin-4 in BALB/c mice with established Leishmania major infections increases the efficacy of antimony therapy and promotes Th1-like responses.

Whereas most inbred mouse strains mount a protective Th1 helper T-cell response following infection with Leishmania major, an ineffective Th2 response develops in BALB/c mice, leading to the development of disseminated, ultimately fatal disease. Interleukin-4 (IL-4) production is required for the initiation of the Th2 response, though little is known about the requirements for the long-term maintenance of this response. In order to investigate the role of the expanding parasite population on the Th2 response, mice infected for 2 weeks with L. major, which exhibited a Th2-like cytokine profile, were treated with a leishmanicidal agent (Pentostam) and/or various doses of anti-IL-4 antibody. Untreated mice, mice treated with Pentostam alone, or mice treated with 2.5 mg of anti-IL-4 antibody given at days 13 and 21 of infection developed progressive disease. However, in 8 of 10 mice treated with this dose of anti-IL-4 antibody plus Pentostam lesion development was arrested and lesions were either controlled or eventually healed. Healing was associated with the production of high levels of gamma interferon by spleen cells, and low levels of immunoglobulin E in serum compared with levels for control animals, indicating that a Th1-like response had developed in mice receiving both treatments. Thus, depletion of IL-4 only in combination with a reduction in the parasite burden allowed the expression of a Th1 response. When the dose of anti-IL-4 antibody was increased to 5 mg per injection, all mice treated with this dose of antibody, with or without Pentostam therapy, healed. However, combined therapy with Pentostam in mice treated with this dose of antibody had an additional protective effect. As expected, a Th1 response developed in mice treated with this dose of anti-IL-4 antibody with or without combined therapy with Pentostam, whereas a Th2 response developed in control mice. Thus, a significant effect on the course of disease is noted when mice with established L. major infections are treated with anti-IL-4 antibody in combination with Pentostam, suggesting that the combined effect of inhibiting IL-4 and reducing the parasite burden has a dramatic effect on the development of resistance to L. major.

Site-specific immunity to Leishmania major in SWR mice: the site of infection influences susceptibility and expression of the antileishmanial immune response.

Inbred strains of mice usually develop either of two divergent patterns of infection in response to Leishmania major. Resistant mice, which develop self-limiting infections, respond immunologically with the activation of gamma interferon-secreting Th1 helper T cells, while nonhealing infections in susceptible mice are characterized by the proliferation of interleukin-4-secreting Th2 cells. Development of these divergent responses is dependent primarily on the strain of mouse infected, although factors such as the infective dose, species, and strain of parasite can also influence the degree of resistance. In this study, we show that a single mouse strain, SWR, can develop totally divergent patterns of infection depending on the site of parasite inoculation. Both SWR mice and highly susceptible BALB/c mice developed progressive, ultimately fatal disease when inoculated in the dorsal skin over the base of the tail. However, SWR mice infected in the hind footpad developed far less severe infections, which were for the most part controlled, whereas BALB/c mice infected in this site developed severe, nonhealing lesions. Production of gamma interferon and interleukin-4 and measurement of immunoglobulin E levels in serum were used to assess the degree of Th1 and Th2 cell activation in infected mice. Cytokine profiles early in infection had characteristics of a mixed Th1-Th2 response and were similar in SWR mice infected at either site. These early cytokine responses were not predictive of the ultimate disease outcome, since lymph node cells from healing mice eventually produced higher levels of gamma interferon than did those from nonhealing mice, and healing mice had lower levels of immunoglobulin E in serum, suggesting a functional bias toward Th1 cell activity in these animals. The differential ability of SWR mice to heal infections at different cutaneous sites provides a new model for the study of resistance to cutaneous leishmaniasis. Unlike traditional models of infection in which resistant and susceptible strains of mice are compared, this model allows for the study of factors that contribute to healing and nonhealing infections in a genetically identical strain of mouse.

Differential control of IFN-gamma and IL-2 production during Trypanosoma cruzi infection.

In murine infection with Trypanosoma cruzi, immune responsiveness to parasite and non-parasite Ag becomes suppressed during the acute phase of infection, and this suppression is known to extend to the production of IL-2. To determine whether suppression of lymphokine production was specific for IL-2, or was a generalized phenomenon involving suppressed production of other lymphokines, we have begun an investigation of the ability of mice to produce of a number of lymphokines during infection, initially addressing this question by studying IFN-gamma production. Supernatants from Con A-stimulated spleen cells from infected resistant (C57B1/6) and susceptible (C3H) mice were assayed for IFN-gamma. Supernatants known to be suppressed with respect to IL-2 production from both mouse strains contained IFN-gamma at or above that of supernatants from normal spleen cells. Samples were assayed in an IFN bioassay to ensure that the IFN-gamma detected by ELISA was biologically active. Thus, suppression during T. cruzi infection does not extend to the production of all lymphokines. The stimulation of IFN-gamma production was confirmed by detection of IFN-gamma mRNA in unstimulated spleen cells from infected animals, and in Con A, Con A + PMA, and in some cases, parasite Ag-stimulated spleen cells from infected animals. IFN-gamma mRNA levels in mitogen-stimulated spleen cells equalled or exceeded those found in similarly stimulated normal cells. In contrast, stimulated spleen cells from infected animals had reduced levels of IL-2 mRNA relative to normal spleen cells. Thus at both the protein and mRNA level, IFN-gamma production is stimulated by T. cruzi infection, whereas IL-2 production is suppressed. Serum IFN-gamma in infected C57B1/6 and C3H mice was detected 8 days after infection, peaked on day 20 of infection, and subsequently fell, but remained detectable at low levels throughout the life of infected mice. Infected animals were depleted of cell populations known to be capable of producing IFN-gamma, and Thy-1+, CD4-, CD8-, NK- cells, and to a lesser degree, CD4+ and CD8+ cells were found to be responsible for the production of IFN-gamma during infection. We also report that IL-2 can induce IFN-gamma production in vitro and in vivo by spleen cells from infected animals, and that IL-2 can synergize with epimastigote or trypomastigote antigen to produce high levels of IFN-gamma comparable to those found in supernatants from mitogen-stimulated cells.