Human Leukocyte Antigens and Cellular Immune Responses to Anthrax Vaccine Adsorbed

Interindividual variations in vaccine-induced immune responses are in part due to host genetic polymorphisms in the human leukocyte antigen (HLA) and other gene families. This study examined associations between HLA genotypes, haplotypes, and homozygosity and protective antigen (PA)-specific cellular immune responses in healthy subjects following immunization with Anthrax Vaccine Adsorbed (AVA). While limited associations were observed between individual HLA alleles or haplotypes and variable lymphocyte proliferative (LP) responses to AVA, analyses of homozygosity supported the hypothesis of a “heterozygote advantage.” Individuals who were homozygous for any HLA locus demonstrated significantly lower PA-specific LP than subjects who were heterozygous at all eight loci (median stimulation indices [SI], 1.84 versus 2.95, P = 0.009). Similarly, we found that class I (HLA-A) and class II (HLA-DQA1 and HLA-DQB1) homozygosity was significantly associated with an overall decrease in LP compared with heterozygosity at those three loci. Specifically, individuals who were homozygous at these loci had significantly lower PA-specific LP than subjects heterozygous for HLA-A (median SI, 1.48 versus 2.13, P = 0.005), HLA-DQA1 (median SI, 1.75 versus 2.11, P = 0.007), and HLA-DQB1 (median SI, 1.48 versus 2.13, P = 0.002) loci, respectively. Finally, homozygosity at an increasing number (≥4) of HLA loci was significantly correlated with a reduction in LP response (P < 0.001) in a dose-dependent manner. Additional studies are needed to reproduce these findings and determine whether HLA-heterozygous individuals generate stronger cellular immune response to other virulence factors (Bacillus anthracis LF and EF) than HLA-homozygous subjects.     

Magnitude of Serum and Intestinal Antibody Responses Induced by Sequential Replicating and Nonreplicating Rotavirus Vaccines in Gnotobiotic Pigs and Correlation with Protection

A sequential mucosal prime-boost vaccine regimen of oral attenuated (Att) human rotavirus (HRV) priming followed by intranasal (i.n.) boosting with rotavirus protein VP2 and VP6 rotavirus-like particles (2/6-VLPs) has previously been shown to be effective for induction of intestinal antibody-secreting cell (ASC) responses and protection in gnotobiotic pigs. Because serum or fecal antibody titers, but not intestinal ASC responses, can be used as potential markers of protective immunity in clinical vaccine trials, we determined the serum and intestinal antibody responses to this prime-boost rotavirus vaccine regimen and the correlations with protection. Gnotobiotic pigs were vaccinated with one of the two sequential vaccines: AttHRV orally preceding 2/6-VLP (VLP2x) vaccination (AttHRV/VLP2x) or following VLP2x vaccination (VLP2x/AttHRV) given i.n. with a mutant Escherichia coli heat-labile toxin (mLT) as adjuvant. These vaccines were also compared with three i.n. doses of VLP+mLT (VLP3x) and one and three oral doses of AttHRV (AttHRV1x and AttHRV3x, respectively). Before challenge all pigs in the AttHRV/VLP2x group seroconverted to positivity for serum immunoglobulin A (IgA) antibodies. The pigs in this group also had significantly higher (P < 0.05) intestinal IgA antibody titers pre- and postchallenge and IgG antibody titers postchallenge compared to those in the other groups. Statistical analyses of the correlations between serum IgM, IgA, IgG, and virus-neutralizing antibody titers and protection demonstrated that each of these was an indicator of protective immunity induced by the AttHRV3x and the AttHRV/VLP2x regimens. However, only IgA and not IgM or IgG antibody titers in serum were highly correlated (R² = 0.89; P < 0.001) with the corresponding isotype antibody (IgA) titers in the intestines among all the vaccinated groups, indicating that the IgA antibody titer is probably the most reliable indicator of protection.     

Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

Previous investigations demonstrated that saponins isolated from the root of Panax ginseng C. A. Meyer (i.e., ginseng root saponin [GS-R]) had adjuvant activity. In the present study, the combined effects of rapeseed oil (RO) and GS-R on the immune responses elicited by foot-and-mouth disease (FMD) vaccine were investigated by measuring FMD virus (FMDV)-specific antibody levels, cytokine levels, lymphocyte proliferation, and long-lived IgG-secreting plasma cells from bone marrow in a mouse model. The results indicated that RO in combination with GS-R significantly enhanced serum IgG and isotype concentrations, gamma interferon (IFN-γ) and interleukin 5 (IL-5) levels, splenocyte proliferative responses to stimulations with concanavalin A (ConA), lipopolysaccharide (LPS), and FMDV antigen, and the numbers of IgG-secreting plasma cells in the bone marrow, suggesting that RO/GS-R enhanced both Th1 and Th2 immune responses. In addition, no significant difference was found between RO/GS-R and the commercial adjuvant oil ISA 206 in the promotion of FMD vaccine-induced immune responses. Considering the vegetable origin of RO and GS-R and the potent adjuvant activity, RO/GS-R should be studied further for the development of veterinary vaccines, especially for use in food animals in order to promote food safety.     

High-Level Cellular and Humoral Immune Responses in Guinea Pigs Immunized Intradermally with a Heat-Inactivated Varicella-Zoster Virus Vaccine

The threat of varicella and herpes zoster in immunocompromised individuals necessitates the development of a safe and effective varicella-zoster virus (VZV) vaccine. The immune responses of guinea pigs to the intradermal (i.d.) or subcutaneous (s.c.) administration of a heat-inactivated or live VZV vaccine were investigated. Relative to nonimmunized animals, a single 399-PFU dose of vaccine induced nonsignificant increases in gamma interferon (IFN-γ), granzyme B, and perforin mRNA expression in the splenocytes of all groups, while two i.d. administrations of the inactivated vaccine increased IFN-γ mRNA expression significantly (P < 0.005). A single 1,995-PFU dose significantly increased the expression of IFN-γ mRNA in the groups receiving the vaccine either i.d. (P < 0.005) or s.c. (P < 0.05), that of granzyme B mRNA in the groups immunized i.d. with the inactivated (P < 0.005) or live (P < 0.005) vaccine, and that of perforin mRNA in the animals that received the inactivated vaccine i.d. (P < 0.005). Importantly, increases in the expression of IFN-γ (P = 0.025), granzyme B (P = 0.004), and perforin (P > 0.05) mRNAs were observed in the animals immunized i.d. with 1,995 PFU of inactivated vaccine relative to those immunized s.c. with the same dose. The proportion of animals expressing IFN-γ mRNA mirrored the proportion expressing IFN-γ protein (correlation coefficient of 0.88). VZV glycoprotein-specific and virus-neutralizing antibodies were produced with no significant intergroup differences. A booster i.d. administration of the 399-PFU dose of heat-inactivated vaccine enhanced the antibody responses. These results demonstrate that i.d. administration of an inactivated VZV vaccine can be an efficient mode of immunization against VZV.     

An Oral versus Intranasal Prime/Boost Regimen Using Attenuated Human Rotavirus or VP2 and VP6 Virus-Like Particles with Immunostimulating Complexes Influences Protection and Antibody-Secreting Cell Responses to Rotavirus in a Neonatal Gnotobiotic Pig Model

We determined the impact of mucosal prime/boost regimens and vaccine type (attenuated Wa human rotavirus [AttHRV] or nonreplicating Wa 2/6 rotavirus-like particles [VLP]) on protection and antibody-secreting cell (ASC) responses to HRV in a neonatal gnotobiotic pig disease model. Comparisons of delivery routes for AttHRV and evaluation of nonreplicating VLP vaccines are important as alternative vaccine approaches to overcome risks associated with live oral vaccines. Groups of neonatal gnotobiotic pigs were vaccinated using combinations of oral (PO) and intranasal (IN) inoculation routes as follows: (i) 3 oral doses of AttHRV (AttHRV3×PO); (ii) AttHRV3×IN; (iii) AttHRVPO, then 2/6VLP2×IN; (iv) AttHRVIN, then 2/6VLP2×IN; and (v) mock-inoculated controls. Subsets of pigs from each group were challenged with virulent Wa HRV [P1A(8) G1] (4 weeks post-primary inoculation) to assess protection. The AttHRVPO+2/6VLP2×IN pigs had the highest protection rates against virus shedding and diarrhea (71% each); however, these rates did not differ statistically among the vaccine groups, except for the AttHRVIN+2/6VLPIN group, which had a significantly lower protection rate (17%) against diarrhea. The isotype, magnitude, and tissue distribution of ASCs were analyzed by enzyme-linked immunospot assay. The highest mean numbers of virus-specific IgG and IgA ASCs were observed pre- and postchallenge in both intestinal and systemic lymphoid tissues of the AttHRVPO+2/6VLPIN group. Thus, the AttHRVPO+2/6VLPIN vaccine regimen using immunostimulating complexes (ISCOM) and multiple mucosal inductive sites, followed by AttHRV3×PO or IN regimens, were the most effective vaccine regimens, suggesting that either AttHRVPO+2/6VLPIN or AttHRV3×IN may be an alternative approach to AttHRV3×PO for inducing protective immunity against rotavirus diarrhea.Safety is a major concern in the use of live vaccines due to potential adverse effects. However, immune responses at mucosal sites are usually most effectively induced in naïve hosts by the administration of living microorganisms. In contrast, nonreplicating vaccines administered orally (PO) require an effective mucosal adjuvant and delivery system to enhance the immunogenicity of the vaccines and to avoid oral tolerance.Rotavirus is the most common cause of infantile gastroenteritis worldwide. Each year, rotavirus causes approximately 25 million clinic visits, 2 million hospitalizations, and 500,000 deaths in children <5 years of age (29). Although two new live attenuated rotavirus (RV) vaccines are licensed, there is an urgent need to develop rotavirus vaccines that are more efficacious in developing countries than the existing vaccines (17, 37). The association of an increased risk of intussusception with the rhesus × human rotavirus reassortant vaccine RotaShield prompted concerns related to possible side effects of live rotavirus vaccines, including excessive virus shedding, fever, diarrhea, vomiting, and irritability, prompting the development of alternative vaccine strategies. Virus-like particles (VLP) composed of rotavirus inner capsid proteins VP2 and VP6 (2/6VLPs) constructed by coexpressing rotavirus gene 2 and gene 6 in a baculovirus expression system using Spodoptera frugiperda (Sf9) insect cells are one strategy (11, 15, 39). However, these 2/6VLP vaccines do not induce rotavirus-neutralizing antibodies. In our previous studies, a vaccine using intranasal (IN) 2/6VLP (2/6VLPIN) (RF VP2, Wa VP6) with mutant Escherichia coli heat-labile toxin (mLT) adjuvant was immunogenic but not protective in gnotobiotic pigs (39). Also, three oral doses of a vaccine of 2/6VLP (2/6VLP3×PO) with immunostimulating complexes (ISCOM) were less immunogenic than three doses of 2/6VLPIN vaccine, but both failed to induce protection against rotavirus diarrhea or shedding (18). However, partial protection against diarrhea and virus shedding was induced when 2/6VLPIN+mLT or 2/6VLPPO+ISCOM was used as a booster vaccine in pigs that were previously primed orally with live attenuated Wa HRV (AttHRVPO+2/6VLPIN+mLT or AttHRVPO+2/6VLPPO+ISCOM) (18, 40). The efficacies of these two vaccine regimens against rotavirus diarrhea (44% and 50%, respectively) were similar or slightly lower than that induced by three oral doses of live attenuated Wa HRV vaccine in pigs (50 to 67%) (18, 40, 41, 43). These results suggest that the prime/boost vaccine regimens may be a more effective approach than multiple doses of either live or VLP vaccines alone.Respiratory symptoms and rotavirus shedding in nasopharyngeal secretions of children have been reported (26, 31), and we previously demonstrated shedding of AttHRV in the respiratory tract (2) of gnotobiotic pigs. Because lymphocytes sensitized in the nasal lymphoid tissue (NALT) can relocate to distant effector sites through the common mucosal immune system (6), the respiratory tract should be explored further as a possible route to improve rotavirus vaccine efficacy. In this study, we first evaluated the dose response to 2/6VLPIN boosting after priming with AttHRVPO. We then assessed the effect of PO versus IN priming with AttHRV using the optimized 2/6VLPIN booster dose. We further compared the impact of IN versus PO priming and boosting using AttHRV alone. Such vaccine regimens, although requiring priming with live AttHRV, may at least reduce the risk associated with live virus boosters or, by using IN delivery, avoid side effects or interference (i.e., intestinal parasites, maternal antibodies, etc.) more prominent in infants in developing countries when live oral rotavirus vaccines are used. Accordingly, the use of 2/6VLP boosters given IN might overcome some of the suppressive effects of maternal antibodies on live oral RV vaccines (19, 25). Bertolotti-Ciarlet et al. (4) showed that oral administration of homologous or heterologous 2/6VLPs to CD-1 mice with or without adjuvant induced low protection rates against rotavirus challenge (10 and 39%), whereas intranasal administration induced higher protection rates (85 and 84%). Their findings together with the findings of others (5) suggest that the harsh environment of the gastrointestinal (GI) tract (3, 34), including the low pH and presence of digestive enzymes which influence the degradation of protein antigens, may account for the differences observed between immunizations using these two routes. Thus, less degradation of VLPs occurs when they are given IN compared to when they are given PO, which may permit lowering of the VLP dosage. The use of an effective and age-appropriate mucosal adjuvant such as ISCOM may also increase the efficacy of the nonreplicating 2/6VLP rotavirus vaccine. Intrarectal immunization was also used to avoid GI degradation. In adult mice, intrarectal immunization using 2/6VLP induced protection against rotavirus shedding and higher intestinal immune responses (1). Similarly, intrarectal inoculation of 8-2/6/7VLP induced complete protection against rotavirus shedding in the same model when administered with LT or cholera toxin (CT) adjuvants (30).The gnotobiotic pig is the only animal model susceptible to HRV diarrhea for at least the first 8 weeks of age, the time necessary to assess protective immunity against disease upon challenge (44), whereas other animal models are susceptible to rotavirus diarrhea only up to 14 to 21 days of age (9, 10). Studies of our selected candidate rotavirus vaccines and new vaccine strategies in neonatal gnotobiotic pigs that mimic immune responses of infants (33) should generate data potentially applicable for the development of similar rotavirus vaccines for infants. Our findings should further improve our understanding of effective prime/boost strategies comparing live versus nonreplicating vaccines and PO versus IN routes to induce intestinal immunity in neonates.(Some data used in Table ​Table22 were partially presented previously, in conjunction with serum and intestinal antibody responses to the AttHRVPO+2/6VLPIN and AttHRV3×PO vaccines, by Gonzalez et al. [16].)

TABLE 2.

Virus shedding and diarrhea in gnotobiotic pigs after challenge with virulent Wa HRV

Immunization with Brucella VirB Proteins Reduces Organ Colonization in Mice through a Th1-Type Immune Response and Elicits a Similar Immune Response in Dogs

VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.     

Intestinal Immune Responses to an Inactivated Oral Enterotoxigenic Escherichia coli Vaccine and Associated Immunoglobulin A Responses in Blood

An inactivated oral enterotoxigenic Escherichia coli (ETEC) vaccine against ETEC diarrhea was given to 25 adult Swedish volunteers. The vaccine consisted of formalin-killed E. coli bacteria expressing the most common colonization factor antigens (CFAs), i.e., CFA/I, -II, and -IV, and recombinantly produced cholera B subunit (CTB). Immunoglobulin A (IgA) antibody responses in intestinal lavage fluid to CTB and CFAs were determined and compared with corresponding responses in stool extracts and serum as well as with IgA antibody-secreting cell (ASC) responses in peripheral blood. Two doses of vaccine induced significant IgA responses to the different CFAs in lavage fluid in 61 to 87% of the vaccinees and in stool in 38 to 81% of them. The most frequent responses were seen against CFA/I. The magnitudes of the antibody responses against CTB and CFA/I in stool correlated significantly (CTB, P < 0.01; CFA/I, P < 0.05) with those in intestinal lavage. Intestinal lavage responses against CFAs were best reflected by the ASC responses, with the sensitivity of the ASC assay being 80 to 85%, followed by stool (sensitivity of 50 to 88%) and serum antibody (sensitivity of 7 to 65%) analyses. CTB-specific immune responses were seen in >90% of the vaccinees in all assays.     

Cholera Toxin B-Subunit Gene Enhances Mucosal Immunoglobulin A, Th1-Type, and CD8+ Cytotoxic Responses When Coadministered Intradermally with a DNA Vaccine

A plasmid vector encoding the cholera toxin B subunit (pCtB) was evaluated as an intradermal genetic adjuvant for a model DNA vaccine expressing the human papillomavirus type 16 L1 capsid gene (p16L1) in mice. p16L1 was coadministered with plasmid pCtB or commercial polypeptide CtB as a positive control. Coadministration of pCtB induced a significant increment of specific anti-L1 immunoglobulin A (IgA) antibodies in cervical secretions (P < 0.05) and fecal extracts (P < 0.005). Additionally, coadministration of pCtB enhanced the production of interleukin-2 and gamma interferon by spleen cells but did not affect the production of interleukin-4, suggesting a Th1-type helper response. Furthermore, improved CD8⁺ T-cell-mediated cytotoxic activity was observed in mice vaccinated with the DNA vaccine with pCtB as an adjuvant. This adjuvant effect was comparable to that induced by the CtB polypeptide. These results indicate that intradermal coadministration of pCtB is an adequate means to enhance the mucosa-, Th1-, and CD8⁺-mediated cytotoxic responses induced by a DNA vaccine.     

Mucosal Immunization with the Live Attenuated Vaccine SPY1 Induces Humoral and Th2-Th17-Regulatory T Cell Cellular Immunity and Protects against Pneumococcal Infection

Mucosal immunization with attenuated vaccine can protect against pneumococcal invasion infection, but the mechanism was unknown. Our study found that mucosal delivery with the live attenuated SPY1 vaccine strain can confer T cell- and B cell-dependent protection against pneumococcal colonization and invasive infection; yet it is still unclear which cell subsets contribute to the protection, and their roles in pneumococcal colonization and invasion remain elusive. Adoptive transfer of anti-SPY1 antibody conferred protection to naive μMT mice, and immune T cells were indispensable to protection examined in nude mice. A critical role of interleukin 17A (IL-17A) in colonization was demonstrated in mice lacking IL-17A, and a vaccine-specific Th2 immune subset was necessary for systemic protection. Of note, we found that SPY1 could stimulate an immunoregulatory response and that SPY1-elicited regulatory T cells participated in protection against colonization and lethal infection. The data presented here aid our understanding of how live attenuated strains are able to function as effective vaccines and may contribute to a more comprehensive evaluation of live vaccines and other mucosal vaccines.     

Humoral and Cell-Mediated Immune Responses to Alternate Booster Schedules of Anthrax Vaccine Adsorbed in Humans

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7, r² = 0.86, P < 0.0001, log₁₀ transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00119067","term_id":"NCT00119067"}}NCT00119067.)     

Identification by Full-Genome Analysis of a Bovine Rotavirus Transmitted Directly to and Causing Diarrhea in a Human Child

The genome of rotaviruses consists of 11 segments of double-stranded RNA, and each genome segment has multiple genotypes. Thus, the genotype constellation of an isolate is often indicative of its host species. Albeit rarely, interspecies transmission occurs either by virions with nonreassorted or reassorted genomic segments. A rotavirus with the G6P[1] genotype, Ro8059, was isolated from the stool of a 1-year-old child during routine characterization of diarrheal specimens from a sentinel clinic in Israel in 1995. Since genotype G6P[1] is generally associated with bovine rotaviruses, and the child developed diarrhea within days of his first contact with calves at an urban farm, the aim of this study was to characterize the whole genomic constellation of Ro8059 and four G6P[1] bovine strains, BRV101, BRV105, BRV106, and CR231/39, by RNA-RNA hybridization and full genome sequencing to determine whether some or all of the segments were of bovine origin. The genome constellations of all four bovine G6P[1] strains were G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3 for VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively. Ro8059 shared the same genotype constellation with these bovine strains, with high nucleotide sequence identities (95.84 to 100%) for each of the 11 segments indicating that Ro8059 represented a direct interspecies whole-genome transmission of a nonreassorted rotavirus from a calf to a human infant. We conclude that this was the earliest example with a complete epidemiological link in which an entirely bovine rotavirus directly infected a human child and caused a symptomatic diarrheal illness. Thus, not all bovine rotaviruses are always naturally attenuated to the human host.     

Immune Responses to Pertussis Antigens in Infants and Toddlers after Immunization with Multicomponent Acellular Pertussis Vaccine

Given the resurgence of pertussis despite high rates of vaccination with the diphtheria-tetanus-acellular pertussis (DTaP) vaccine, a better understanding of vaccine-induced immune responses to Bordetella pertussis is needed. We investigated the antibody, cell-mediated, and cytokine responses to B. pertussis antigens in children who received the primary vaccination series (at 2, 4, and 6 months) and first booster vaccination (at 15 to 18 months) with 5-component acellular pertussis (aP) vaccine. The majority of subjects demonstrated a 4-fold increase in antibody titer to all four pertussis antigens (pertussis toxin [PT], pertactin [PRN], filamentous hemagglutinin [FHA], and fimbriae [FIM]) following the primary series and booster vaccination. Following the primary vaccine series, the majority of subjects (52 to 67%) mounted a positive T cell proliferative response (stimulation index of ≥3) to the PT and PRN antigens, while few subjects (7 to 12%) mounted positive proliferative responses to FHA and FIM. One month after booster vaccination (age 16 to 19 months), our study revealed significant increase in gamma interferon (IFN-γ) production in response to the PT and FIM antigens, a significant increase in IL-2 production with the PT, FHA, and PRN antigens, and a lack of significant interleukin-4 (IL-4) secretion with any of the antigens. While previous reports documented a mixed Th1/Th2 or Th2-skewed response to DTaP vaccine in children, our data suggest that following the first DTaP booster, children aged 16 to 19 months have a cytokine profile consistent with a Th1 response, which is known to be essential for clearance of pertussis infection. To better define aP-induced immune responses following the booster vaccine, further studies are needed to assess cytokine responses pre- and postbooster in DTaP recipients.     

Role of Th1 and Th2 Cytokines in Immune Response to Uncomplicated Plasmodium falciparum Malaria

The relative balance between Th1 and Th2 cytokines appears crucial, since the role of cytokines has been evaluated in several studies by comparison of clinically heterogeneous groups of patients. The aim of this study is to determine the role of proinflammatory Th1 cytokines, interleukin-12 (IL-12) and gamma interferon (IFN-γ), and anti-inflammatory Th2 cytokines, IL-4 and IL-10, in a homogeneous group of patients with uncomplicated Plasmodium falciparum malaria. Levels of IL-12, IFN-γ, Il-4, and IL-10 in serum for 20 adult patients and 15 healthy control subjects were determined by an immunoenzymatic assay. Serum levels of Th1 cytokines, IL-12 (8.6 ± 2.8 pg/ml; controls, 3.2 ± 0.7 pg/ml) and IFN-γ (39.2 ± 67.6 pg/ml; controls, 8.4 ± 6.3 pg/ml), were significantly increased at admission; 3 days later, levels of IL-12 in serum remained significantly high (8.8 ± 2.6 pg/ml), whereas IFN-γ levels returned to control values. The anti-inflammatory response of Th2 cytokines (IL-10 and IL-4) was distinct. Levels of IL-10 in serum were not significantly increased at day 0 and day 3 (306.6 ± 200.4 pg/ml and 56.6 ± 38.4 pg/ml, respectively; controls, 17.4 ± 9.0 pg/ml). In contrast, levels of IL-4 in serum were not increased on admission (3.4 ± 1.2 pg/ml; controls, 2.4 ± 0.8 pg/ml), but at day 3 a moderate and significant increase of IL-4 levels was observed (4.5 ± 1.7 pg/ml). In conclusion, the increase of Th1 cytokine IL-12 and IFN-γ levels during the acute phase of uncomplicated P. falciparum malaria may reflect an early and effective immune response regulated by proinflammatory Th1 cytokines, and in particular IFN-γ may play a role in limiting progression from uncomplicated malaria to severe and life-threatening complications.     

Elevated Interleukin-21 Correlated to Th17 and Th1 Cells in Patients with Immune Thrombocytopenia

Background  

Interleukin-21 (IL-21) is critical in the development of autoimmune diseases. The role of IL-21 in the pathogenesis of immune thrombocytopenia (ITP) remains unknown.     

Human Monocyte-Derived Dendritic Cells Exposed to Microorganisms Involved in Hypersensitivity Pneumonitis Induce a Th1-Polarized Immune Response

Hypersensitivity pneumonitis (HP) is an immunoallergic disease characterized by a prominent interstitial infiltrate composed predominantly of lymphocytes secreting inflammatory cytokines. Dendritic cells (DCs) are known to play a pivotal role in the lymphocytic response. However, their cross talk with microorganisms that cause HP has yet to be elucidated. This study aimed to investigate the initial interactions between human monocyte-derived DCs (MoDCs) and four microorganisms that are different in nature (Saccharopolyspora rectivirgula [actinomycetes], Mycobacterium immunogenum [mycobacteria], and Wallemia sebi and Eurotium amstelodami [filamentous fungi]) and are involved in HP. Our objectives were to determine the cross talk between MoDCs and HP-causative agents and to determine whether the resulting immune response varied according to the microbial extract tested. The phenotypic activation of MoDCs was measured by the increased expression of costimulatory molecules and levels of cytokines in supernatants. The functional activation of MoDCs was measured by the ability of MoDCs to induce lymphocytic proliferation and differentiation in a mixed lymphocytic reaction (MLR). E. amstelodami-exposed (EA) MoDCs expressed higher percentages of costimulatory molecules than did W. sebi-exposed (WS), S. rectivirgula-exposed (SR), or M. immunogenum-exposed (MI) MoDCs (P < 0.05, Wilcoxon signed-rank test). EA-MoDCs, WS-MoDCs, SR-MoDCs, and MI-MoDCs induced CD4⁺ T cell proliferation and a Th1-polarized immune response. The present study provides evidence that, although differences were initially observed between MoDCs exposed to filamentous fungi and MoDCs exposed to bacteria, a Th1 response was ultimately promoted by DCs regardless of the microbial extract tested.     

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8⁺ T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8⁺ T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4⁺ T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8⁺ T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8⁺ T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D^b-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 10⁵ TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.Cancers pose unique challenges to therapeutic vaccines. Tumor-associated antigens are often self-antigens to which the patient is tolerant. In the case of virus-associated tumors, the viral oncoproteins commonly lack high-avidity T-cell epitopes and thus can evade immune surveillance. Cancer patients frequently show immunological abnormalities, such as T-cell anergy, peripheral and central tolerance, regulatory T cell (Treg)-mediated immunosuppression, and functional T-cell exhaustion (9, 31, 36). Therapeutic cancer vaccines, unlike prophylactic vaccines, thus need to be formulated not only to induce T-cell responses but also to overcome immunological unresponsiveness to tumor antigens.Cervical cancer is the second most common cause of cancer death in women, claiming approximately 400,000 to 500,000 lives each year worldwide (32). Cervical cancer affects ∼1% of all women and is the most common cause of cancer death in women under the age of 50. Virtually all cases of cervical cancers are associated with human papillomavirus (HPV) infections (2, 37). Prevalence of sexually transmitted infections with oncogenic genotypes of HPV varies from 20 to 80% of sexually active adults depending on the study population, with HPV type 16 (HPV-16) representing the most epidemiological relevant oncogenic virus type (2, 37). Two vaccines that express the major capsid protein-inducing serotype-specific HPV neutralizing antibodies have recently become available for preventive vaccination (14). While these vaccines can prevent virus infections with the corresponding HPV genotypes, they cannot affect viral clearance in already infected women or inhibit the development of HPV-associated malignancies. In contrast, therapeutic vaccines targeting HPV oncoproteins, mainly E6 and E7, which are expressed by all transformed epithelial cells, can activate antigen-specific cytotoxic CD8⁺ T-cell responses and eradicate infected cells before or after the malignant transformation event (18, 21, 27). In contrast to the conventional vaccines, such oncoprotein-based anti-HPV vaccine formulations may be used under either preventive or therapeutic conditions regarding the establishment and uncontrolled growth of the cancer cells under both experimental or clinical conditions (18).Although DNA vaccines have been intensively studied as a promising immunization strategy for the control of HPV-associated tumors, vectors expressing HPV-16 E7 or E6 oncoprotein alone have shown low antigen-specific CD8⁺ T-cell activation and lack of protective antitumor effects in mice (5, 21, 30). Improved activation of antigen-specific CD8⁺ T-cell responses by anti-HPV DNA vaccines were achieved after genetic fusion of the E7 or E6 oncoprotein with different carrier proteins carrying cell targeting signals or mediators of immune responses (8-19). The focus of our DNA vaccines targeting HPV-induced cancers has been the augmentation of adaptive immune responses through the blockade of an immune inhibitory pathway based on the expression of hybrid proteins genetically fused with glycoprotein D (gD) of herpes simplex virus (HSV) (24,25). HSV gD binds the herpes virus entry mediator (HVEM) and competes for the same binding site as the B- and T-lymphocyte attenuator (BTLA). BTLA provides inhibitory signals to T and B cells upon binding to HVEM (7, 38). Blockade of the HVEM-BTLA pathway during activation of an adaptive immune response has been associated with increased immune responses, particularly E7-specific CD8⁺ T-cell responses, to the antigen encoded by the DNA vaccine (25).We reported previously that expression of HPV-16 E5, E6, and E7 oncoproteins within HSV type 1 (HSV-1) gD, either by an adenovirus vector or a DNA vaccine, induces a potent CD8⁺ T-cell response that confers preventive protection to mice challenged with transplantable E6- and E7-expressing TC-1 cells (25). Herein we characterized previously unknown aspects of the antigen-specific immune responses elicited in mice immunized with the DNA vaccine and report, for the first time, the therapeutic antitumor effects of the pgD-E7E6E5 vector in mice. The results indicate that the DNA vaccine encoding chimeric oncoproteins genetically fused to the HSV-1 gD protein represents a promising approach for the therapeutic control of HPV-associated tumors.     

Genetic Control of Immune Response to Recombinant Antigens Carried by an Attenuated Salmonella typhimurium Vaccine Strain: Nramp1 Influences T-Helper Subset Responses and Protection against Leishmanial Challenge

Attenuated strains of Salmonella typhimurium have been widely used as vehicles for delivery and expression of vaccine antigens in murine models of infectious disease. In mice, early bacterial replication following infection with S. typhimurium is controlled by the gene (Nramp1, formerly Ity/Lsh/Bcg) encoding the natural-resistance-associated macrophage protein (Nramp1). Nramp1 regulates macrophage activation and has multiple pleiotropic effects, including regulation of tumor necrosis factor alpha, interleukin 1β (IL-1β), and major histocompatibility complex class II molecules, all of which influence antigen processing and presentation. Nramp1 also has a direct effect on antigen processing, possibly by regulating the activity of proteases in the late endosomal compartment. Hence, there are multiple ways (regulation of bacterial load or recombinant antigen dose, class II molecule expression, costimulatory or adjuvant activity, and antigen processing) that Nramp1 might influence responses to recombinant salmonella vaccines. To test the hypothesis that Nramp1 influences responses to vaccination, congenic mouse strains have been used to analyze immune responses to recombinant antigens (tetanus toxoid antigen and leishmanial gp63) carried by live attenuated S. typhimurium aroA aroD mutants. Results show that congenic mice carrying the wild-type (S. typhimurium resistance) Nramp1 allele mount a predominantly T-helper-1 (IL-2 and gamma interferon) response to vaccination and show enhanced resolution of lesions following challenge infection with Leishmania major. In contrast, mice carrying mutant (S. typhimurium susceptibility) Nramp1 mount a T-helper-2 (immunoglobulin E and IL-4) response and show exacerbated lesion growth upon challenge.Attenuated strains of Salmonella have been widely used as vehicles for delivery and expression of a heterogeneous range of antigens (Ag) from other pathogens (for a review, see reference 18). In murine models of infectious disease, genetically engineered Salmonella typhimurium has been used to induce immunity to viral (influenza virus A [40]), bacterial (tetanus toxin [10], Streptococcus pyogenes M1 protein [31], and Francisella tularensis [36]), protozoan (Leishmania major [44, 45] and Plasmodium yoelii circumsporozoite protein [33]), and helminth (schistosomiasis [22, 23]) Ag. Since a similar vaccine strategy (e.g., attenuated Salmonella typhi aro mutants in human trials [16, 39]) might ultimately be adopted for use in genetically diverse human populations, it is important to investigate the influence that host genetics might have on the ability to induce protective immune responses to recombinant salmonella vaccines. While major histocompatibility complex (MHC) class I and class II molecule genes with polymorphisms will be obvious candidate genes because of their ability to restrict vaccine responses to certain antigenic epitopes, a role for non-MHC genes acting independently of Ag specificity should also be considered.In mice, early bacterial replication following infection with S. typhimurium is regulated by the gene (Nramp1, formerly Ity/Lsh/Bcg) encoding the natural-resistance-associated macrophage protein (Nramp1) (for reviews, see references 4, 6, and 37). Nramp1 was identified by positional cloning (42). Gene knockout was then used (41) to formally demonstrate that the gene (renamed Nramp1) mediated resistance to all three intramacrophage pathogens S. typhimurium (Ity), Leishmania donovani (Lsh), and Mycobacterium bovis (Bcg). The protein (Nramp1) encoded by Nramp1 regulates the cascade of gene-inductive events which follow interaction of macrophages with bacterial lipopolysaccharide (LPS) and/or natural killer- or T-cell-derived gamma interferon (IFN-γ). The gene has multiple pleiotropic effects, including regulation of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and MHC class II molecules. All of these influence Ag processing and presentation, either directly (class II) or indirectly through their costimulatory or adjuvant (IL-1β and TNF-α) activity. Recent studies also demonstrate that Nramp1 has a more direct effect on Ag processing, possibly by regulating the activity of proteases in the late endosomal compartment (26). Hence, there are multiple ways (regulation of bacterial load or recombinant Ag dose, class II molecule expression, costimulatory or adjuvant activity, and Ag processing) that Nramp1 might influence responses to recombinant salmonella vaccines.To test the hypothesis that Nramp1 influences responses to vaccination, congenic mouse strains have been used to analyze immune responses to recombinant Ag (tetanus toxoid Ag and leishmanial gp63) carried by live attenuated S. typhimurium aroA aroD mutants. Results show that congenic mice carrying the wild-type (S. typhimurium resistance) Nramp1 allele mount a predominantly T-helper-1 (IL-2 and IFN-γ) response to vaccination and show enhanced resolution of lesions following challenge infection with L. major. In contrast, mice carrying mutant (S. typhimurium susceptibility) Nramp1 mount a T-helper-2 (immunoglobulin E [IgE] and IL-4) response and show exacerbated lesion growth upon challenge.