Humoral immunity to smallpox vaccines and monkeypox virus challenge: proteomic assessment and clinical correlations.

Despite the eradication of smallpox, orthopoxviruses (OPV) remain public health concerns. Efforts to develop new therapeutics and vaccines for smallpox continue through their evaluation in animal models despite limited understanding of the specific correlates of protective immunity. Recent monkeypox virus challenge studies have established the black-tailed prairie dog (Cynomys ludovicianus) as a model of human systemic OPV infections. In this study, we assess the induction of humoral immunity in humans and prairie dogs receiving Dryvax, Acam2000, or Imvamune vaccine and characterize the proteomic profile of immune recognition using enzyme-linked immunosorbent assays (ELISA), neutralization assays, and protein microarrays. We confirm anticipated similarities of antigenic protein targets of smallpox vaccine-induced responses in humans and prairie dogs and identify several differences. Subsequent monkeypox virus intranasal infection of vaccinated prairie dogs resulted in a significant boost in humoral immunity characterized by a shift in reactivity of increased intensity to a broader range of OPV proteins. This work provides evidence of similarities between the vaccine responses in prairie dogs and humans that enhance the value of the prairie dog model system as an OPV vaccination model and offers novel findings that form a framework for examining the humoral immune response induced by systemic orthopoxvirus infection.

Establishment of the black-tailed prairie dog (Cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high- and low-dose monkeypox virus challenges.

The 2003 monkeypox virus (MPXV) outbreak and subsequent laboratory studies demonstrated that the black-tailed prairie dog is susceptible to MPXV infection and that the ensuing rash illness is similar to human systemic orthopoxvirus (OPXV) infection, including a 7- to 9-day incubation period and, likely, in some cases a respiratory route of infection; these features distinguish this model from others. The need for safe and efficacious vaccines for OPVX in areas where it is endemic or epidemic is important to protect an increasingly OPXV-naïve population. In this study, we tested current and investigational smallpox vaccines for safety, induction of anti-OPXV antibodies, and protection against mortality and morbidity in two MPXV challenges. None of the smallpox vaccines caused illness in this model, and all vaccinated animals showed anti-OPXV antibody responses and neutralizing antibody. We tested vaccine efficacy by challenging the animals with 10(5) or 10(6) PFU Congo Basin MPXV 30 days postvaccination and evaluating morbidity and mortality. Our results demonstrated that vaccination with either Dryvax or Acambis2000 protected the animals from death with no rash illness. Vaccination with IMVAMUNE also protected the animals from death, albeit with (modified) rash illness. Based on the results of this study, we believe prairie dogs offer a novel and potentially useful small animal model for the safety and efficacy testing of smallpox vaccines in pre- and postexposure vaccine testing, which is important for public health planning.

Immune aspects of Bartonella.

Bartonella species have been recognized as important human pathogens only recently. Until the early 1990s, this genus was represented by one species, Bartonella bacilliformis. The recent identification of other Bartonella species as the agents of cat-scratch disease and bacillary angiomatosis has left little doubt of their emerging importance as opportunistic human pathogens. Over the last decade, extensive research has been performed on Bartonella species, resulting in an explosion in our knowledge of the genetic diversity of this genus. Unusual aspects of disease sequelae have fueled worldwide interest in defining the natural history, pathology, and molecular biology of Bartonella species. While much information about these interests has been presented, the advancement of immunological knowledge regarding Bartonella species has been slow. This review discusses immunological data on Bartonella species, focusing on the three primary human pathogens of this genus: B. bacilliformis, B. quintana, and B. henselae.

Bartonella henselae, B. quintana, and B. bacilliformis: historical pathogens of emerging significance.

Bartonella species were virtually unrecognized as modern pathogens of humans until the last decade. However, identification of Bartonella species as the agents of cat-scratch disease, bacillary angiomatosis, urban trench fever, and possible novel presentations of Carrion's disease has left little doubt of the emerging medical importance of this genus of organisms. The three primary human pathogenic bartonellae, Bartonella bacilliformis (Carrion's disease), B. henselae (cat-scratch disease), and B. quintana (trench fever), present noteworthy comparisons in the epidemiology, natural history, pathology, and host-microbe interaction that this review will briefly explore.

Characterization of Bartonella henselae-specific immunity in BALB/c mice.

BALB/c mice were inoculated with Bartonella henselae by both systemic and mucosal routes. Culture analysis of tissues from mice infected intraperitoneally with a high dose of B. henselae yielded positive results 24 hr after infection. However, culture analysis of blood taken between 6 hr and 7 days after infection from groups receiving live B. henselae were negative. Following intraperitoneal infection, B. henselae was detected by polymerase chain reaction in liver and mesenteric lymph nodes by 6 hr and up to 7 days after infection in liver, kidney and spleen tissue. Enzyme-linked immunosorbent assay (ELISA) of serum samples collected as early as 13 days after infection indicated humoral immune responses to B. henselae. Specific humoral responses remained through week 6. Analysis of faecal samples revealed induction of B. henselae-specific immunoglobulin A by day 28 after infection. In addition, B. henselae-specific cellular responses were indicated by a positive delayed-type hypersensitivity and a T helper 1 (Th1) (CD4+ T cell)-type cytokine response following in vitro stimulation of splenocytes. The significance and implications of these data in relation to B. henselae infections are discussed.

Characterization of human immunoglobulin (Ig) isotype and IgG subclass response to Bartonella henselae infection.

Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera against Rickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintana antigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly to Bartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection with B. henselae or B. quintana.

Protection by minigenes: a novel approach of DNA vaccines.

To test the principle that genetically engineered epitopes in a plasmid DNA can efficiently induce specific immunity, a minigene cassette encoding cytotoxic T lymphocyte (CTL), helper T and B cell epitopes from herpes simplex virus (HSV) was constructed and placed in an expression vector named pcMini. Following immunizations with pcMini, mice developed epitope-specific CTLs comparable to the response induced by live HSV. Less effective but detectable antibody, lymphoproliferation, and T cell cytokine responses were also produced. In addition, pcMini-primed mice elicited a recall response upon restimulation with recombinant vaccinia virus expressing HSV antigen. The protection provided by minigene vaccination was significant, although not as efficient as live virus vaccine. The DNA minigene approach may prove useful to define and induce immune responses against minimal antigenic determinants.

Protective immunity against herpes simplex virus (HSV) type 1 following oral administration of recombinant Salmonella typhimurium vaccine strains expressing HSV antigens.

Salmonella typhimurium strains expressing foreign antigens of various pathogens are capable of eliciting antigen-specific humoral and cellular immune responses. Attenuated S. typhimurium strain chi4550 (delta(cya) delta(crp) delta(asd)) was used as an expression vector for herpes simplex virus (HSV) antigens. Genes encoding glycoprotein D (gD) and the immediate-early protein ICP27 of HSV-1 were cloned and expressed in plasmid pYA292 (asd+) and subsequently placed into chi4550. Following two oral immunizations, the protective efficacy of recombinant strains against zosteriform challenge with HSV-1 was measured in 3-4-week-old BALB/c mice. Levels of protection observed were 77% with the ICP27 construct but only 31% with the gD construct. Zosteriform protection correlates with a CD4+-mediated delayed-type hypersensitivity (DTH) reaction against HSV. Accordingly, significant DTH was observed only in mice immunized orally with the S. typhimurium ICP27 construct. ELISA analysis of antigen-specific humoral responses failed to detect serum antibody responses following oral administration although recombinant S. typhimurium were isolated from spleens of orally dosed mice up to day 30. Intravenous (i.v.) immunization with the gD-expressing construct did, however, induce detectable serum antibody responses. Some humoral IgA responses against gD in faecal samples were detected as early as 3 weeks post-oral immunization while those induced by the i.v. route were slightly lower. These data suggest that recombinant S. typhimurium HSV antigens are capable of inducing immunity against HSV, some aspects of which are protective against HSV challenge.

DNA vaccines -- a modern gimmick or a boon to vaccinology?

The reports in 1993 that naked DNA encoding viral genes conferred protective immunity came as a surprise to most vaccinologists. This review analyses the expanding number of examples where plasmid DNA induces immune responses. Issues such as the type of immunity induced, mechanisms of immune protection, and how DNA vaccines compare with other approaches are emphasized. Additional issues discussed include the likely means by which DNA vaccines induce CTL, how the potency and type of immunity induced can be modified, and whether DNA vaccines represent a practical means of manipulating unwanted immune response occurring during immunoinflammatory diseases. It seems doubtful if DNA vaccines will replace currently effective vaccines, but they may prove useful for prophylactic use against some agents that at present lack an effective vaccine. DNA vaccines promise to be valuable to manipulate the immune response in situations where responses to agents are inappropriate or ineffective.

Cytokine expression in the gut associated lymphoid tissue after oral administration of attenuated Salmonella vaccine strains.

The use of attenuated Salmonella vaccine vectors as delivery vehicles for heterologous antigens has been extensive. The majority of work has analyzed the specific immune responses to the recombinant antigen in question. In addition, most analysis has been performed on animals maintained with sterile food, water, and bedding. This report defines the Salmonella specific cytokine responses in the gut associated lymphoid tissue after oral immunization with two highly publicized attenuated Salmonella carrier strains in animals maintained with nonsterile food, water, and bedding. Increases in expression of both IL-4 and IFN-gamma occur following immunization with either Salmonella construct. In addition, other cytokines (IL-6, IL-7, IL-12) increase at similar levels in either BRD509 or KR1 dosed animals. Proinflammatory cytokines IL-1 and TNF-alpha are also present but unchanged at early time points (6, 24, and 72 hours), increasing only after 7 days postimmunization. These data have implications in the generation of immunity to recombinant antigens since the response to Salmonella will dictate the direction of responses in terms of CD4 T cell activation.

Differential induction of carrier antigen-specific immunity by Salmonella typhimurium live-vaccine strains after single mucosal or intravenous immunization of BALB/c mice.

In this study, we constructed strain KR21 (chi 4550 delta cya delta crp delta asd/pYA292asd(+)-toxC+) and compared it with BRD847 (aroA aroD/pnirB-toxC) for the ability to induce humoral and cellular immunity after a single oral or intravenous immunization in 3- to 4-week-old BALB/c mice. ToxC-specific serum immunoglobulin G (IgG) was detectable in animals orally immunized with either BRD847 or KR21. However, after intravenous immunization, IgG was detected only in BRD847-immunized animals. Measurement of immunoglobin types IgG1 and IgG2a suggests that a Th1 cellular response is prominent after immunizations with either system. ToxC-specific IgA was detected in fecal and vaginal samples of animals immunized orally and intravenously with BRD847, while those immunized with KR21 failed to show fecal or vaginal IgA responses. Delayed-type hypersensitivity was used as a measure of induction of T-cell responses in vivo. Mice immunized either orally or intravenously with BRD847 showed significant ear swelling responses after ToxC injections, while KR21-immunized animals failed to show a cellular response. These data indicate that the aroA aroD/pnirB system holds greater potential for inducing global immunity after a single dose when directly compared with the balanced lethal system (delta cya delta crp delta asd/pYA292asd+).

Adaptive acid tolerance response (ATR) in Aeromonas hydrophila.

Aeromonas hydrophila, a gastrointestinal pathogen of humans, was shown to exhibit a significant adaptive acid tolerance response (ATR) capable of protecting cells from severe acid at a pH of 3.5. The ATR was induced by exposure to a relatively mild pH level of 5.0 for 20 min. Adaptation required protein synthesis since treatment with chloramphenicol during adaptation to pH 5.0 prevented the development of acid tolerance. The adaptation to acid environment was found to be a non-transient phenomenon. Also, iron was not required for acid adaptation in A. hydrophila. Two-dimensional protein analyses revealed an increased production of 28 proteins and decreased synthesis of 10 following pH shifts from 7.2 to 5.0. The mild pH treatment must act as a signal to A. hydrophila to adapt and survive in acid environments by producing 'protective' proteins. The adaptation and survival of this pathogen in low pH may provide valuable information about its ability to withstand acid environments in nature and in the human gastrointestinal tract.