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OBJECTIVES/GOALS: We prioritize Chlamydia’s public health impact, aim to develop rVCG-MECA for practical use, study robust immunity for effective strategies, and assess animal models for human vaccination adaptation. Our work highlights rVCG-MECA’s translational significance in public health. METHODS/STUDY POPULATION: Female Mice C57BL/6J mice (N=8) were immunized intramuscularly(IM) and intranasally(IN) and boosted twice, two weeks apart, with rVCG-MECA, once with live Chlamydia (C. trachomatis serovar D elementary bodies) and PBS. Specific mucosal and systemic immune responses were characterized. Vaccine efficacy was determined from chlamydia shedding following the transcervical challenge. Additionally, Chlamydia-specific cytokine (IFN-γ and IL-4) production by splenic and ILN T cells was assessed after 16 weeks RESULTS/ANTICIPATED RESULTS: Immunization with rVCG-MECA via intramuscular and intranasal routes triggered notable humoral responses in systemic and mucosal tissues. Intramuscular vaccination produced higher IgG2c levels in both tissues, while intranasal vaccination led to elevated IgA levels in mucosal tissues. rVCG-MECA-immunized mice exhibited significantly higher IFN-γ (Th1) secretion compared to IL-4 (Th2), with intramuscular immunization showing the highest IFN-γ levels. These findings anticipate robust immune responses, promising protection against Chlamydia, particularly through the intra muscular route. Overall, our results support rVCG-MECA as a promising Chlamydia vaccine, aligned with public health goals. DISCUSSION/SIGNIFICANCE: This study suggests that IM and IN immunization with rVCG-MECA induces immune effectors such as IFN-gamma and IgG2c that mediate chlamydial clearance in the genetical tract.

Chlamydia abortus (Cab) causes spontaneous abortion and neonatal mortality in infected ruminants and pregnant women. Most Cab infections are asymptomatic, although they can be treated with antibiotics, signifying that control of these infections may require alternative strategies, including the use of effective vaccines. However, the limitations imposed by live attenuated and inactivated vaccines further suggest that employment of subunit vaccines may need to be considered. The efficacy of a newly generated Vibrio cholerae ghost (rVCG)-based subunit vaccine harboring the N-terminal portion of the Cab Pmp18D protein (rVCG-Pmp18.3) in preventing Cab-induced abortion or neonatal mortality was evaluated in pregnant mice. Mice were intranasally (IN) immunized and boosted twice, 2 weeks apart with the vaccine, and immunized and unimmunized mice were caged with males 4 weeks postimmunization. The mice were then infected either IN or transcervically (TC) 10 days after pregnancy, and the fertility rate was determined 7 days postpartum. Eight days after delivery, the mice were sacrificed, and Cab infectivity in the lungs and spleens was evaluated by culturing tissue homogenates in tissue culture. Our results demonstrated that the vaccine induced immune effectors that mediated complete clearance of infection in the lungs and significantly reduced Cab infectivity in the spleen following IN immunization. Vaccine immunization also afforded protection against Cab-induced upper genital tract pathology (uterine dilation). Furthermore, while there was no incidence of abortion in both immunized and unimmunized mice, immunized mice were completely protected against neonatal mortality compared to unimmunized infected controls, which lost a significant percentage of their litter 7 days postpartum. Our results establish the capability of the rVCG-Pmp18.3 vaccine to prevent infection in the lungs (mucosal) and spleen (systemic) and protect mice from Cab-induced tubal pathologies and neonatal mortality, a hallmark of Cab infection in ruminants. To advance the commercial potential of this vaccine, future studies will optimize the antigen dose and the number of vaccine doses required for protection of ruminants. [ABSTRACT FROM AUTHOR]

We have previously shown that the time of Chlamydia infection was crucial in determining the chlamydial infectivity and pathogenesis. This study aims to determine whether the time of Chlamydia infection affects the genital tract microbiome. This study analyzed mice vaginal, uterine, and ovary/oviduct microbiome with and without Chlamydia infection. The mice were infected with Chlamydia at either 10:00 am (ZT3) or 10:00 pm (ZT15). The results showed that mice infected at ZT3 had higher Chlamydia infectivity than those infected at ZT15. There was more variation in the compositional complexity of the vaginal microbiome (alpha diversity) of mice infected at ZT3 than those mice infected at ZT15 throughout the infection within each treatment group, with both Shannon and Simpson diversity index values decreased over time. The analysis of samples collected four weeks post-infection showed that there were significant taxonomical differences (beta diversity) between different parts of the genital tract—vagina, uterus, and ovary/oviduct—and this difference was associated with the time of infection. Firmicutes and Proteobacteria were the most abundant phyla within the microbiome in all three genital tract regions for all the samples collected during this experiment. Additionally, Firmicutes was the dominant phylum in the uterine microbiome of ZT3 Chlamydia infected mice. The results show that the time of infection is associated with the microbial dynamics in the genital tract. And this association is more robust in the upper genital tract than in the vagina. This result implies that more emphasis should be placed on understanding the changes in the microbial dynamics of the upper genital tract over the course of infection. [ABSTRACT FROM AUTHOR]

Abstract Background Resistance to sulphadoxine-pyrimethamine (SP) in Plasmodium falciparum parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes and has spread worldwide. SP remains the recommended drug for intermittent preventive treatment for malaria in pregnancy (IPTp) and information on population prevalence of the SP resistance molecular markers in pregnant women is limited. Methods Temporal trends of SP resistance molecular markers were investigated in 489 parasite samples collected from pregnant women at delivery from three different observational studies between 1996 and 2009 in Kenya, where SP was adopted for both IPTp and case treatment policies in 1998. Using real-time polymerase chain reaction, pyrosequencing and direct sequencing, 10 single-nucleotide polymorphisms (SNPs) of SP resistance molecular markers were assayed. Results The prevalence of quintuple mutant (dhfr N51I/C59R/S108N and dhps A437G/K540E combined genotype) increased from 7 % in the first study (1996–2000) to 88 % in the third study (2008–2009). When further stratified by sample collection year and adoption of IPTp policy, the prevalence of the quintuple mutant increased from 2.4 % in 1998 to 44.4 % three years after IPTp policy adoption, seemingly in parallel with the increase in percentage of SP use in pregnancy. However, in the 1996–2000 study, more mutations in the combined dhfr/dhps genotype were associated with SP use during pregnancy only in univariable analysis and no associations were detected in the 2002–2008 and 2008–2009 studies. In addition, in the 2008–2009 study, 5.3 % of the parasite samples carried the dhps triple mutant (A437G/K540E/A581G). There were no differences in the prevalence of SP mutant genotypes between the parasite samples from HIV + and HIV- women over time and between paired peripheral and placental samples. Conclusions There was a significant increase in dhfr/dhps quintuple mutant and the emergence of new genotype containing dhps 581 in the parasites from pregnant women in western Kenya over 13 years. IPTp adoption and SP use in pregnancy only played a minor role in the increased drug-resistant parasites in the pregnant women over time. Most likely, other major factors, such as the high prevalence of resistant parasites selected by the use of SP for case management in large non-pregnant population, might have contributed to the temporally increased prevalence of SP resistant parasites in pregnant women. Further investigations are needed to determine the linkage between SP drug resistance markers and efficacy of IPTp-SP.

Abstract Background MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP119), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP119 had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP119 would affect critical T-cell responses to epitopes in this antigen. Methods The cellular responses to wild-type MSP119 and a panel of modified MSP119 antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults. Results Interestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP119. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins. Conclusion This study suggests that specific MSP119 variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.