Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

To determine the role of maternal immunity in protecting newborn mice against a C. trachomatis infection female BALB/c mice were immunized intranasally (i.n.) with 104 inclusion forming units (IFU) of the C. trachomatis mouse pneumonitis biovar (MoPn). As a control, another group of female mice was sham-immunized i.n. with HeLa cell extracts. Immunized animals mounted strong immune responses as evidenced by high Chlamydia-specific antibody titers in serum and milk. Newborn mice born from immunized and sham-immunized dams were challenged i.n. with 103 IFU of MoPn at 2-post natal days (PND). Following inoculation, newborn mice were euthanized at 7-PND and 18-PND and the lungs, spleen and intestine were cultured for Chlamydia. Overall, no significant differences were observed between the mice born from and fed by immunized dams and mice born from and fed by sham-immunized dams. Of the mice born from immunized dams, 75% and 25% had positive lung cultures at 7-PND and 18-PND, respectively. Of the mice born from sham-immunized dams, 82% and 50% had positive lung cultures for those same days. When the number of IFU recovered from the lungs and spleens were compared between the two groups no significant differences were observed. However, when the number of IFU recovered from the small intestine were compared, significant differences were observed between the two groups of newborn mice (2×105 versus 32×106 at 7-PND and 9.2×106 versus 85×106 at 18-PND). In conclusion, maternal immunity plays a limited role in protecting newborn mice against a Chlamydia infection.

SummaryC3H/HeN female mice were vaccinated with native Chlamydia muridarum major outer membrane protein (MOMP), using Montanide+CpG or Alum+CpG as adjuvants. Negative control groups were immunized with ovalbumin (OVA) and the same adjuvants. As positive control, mice were inoculated intranasally with live Chlamydia. Mice were challenged in the ovarian bursa with 10 5 C. muridarum inclusion forming units. Six weeks after the genital challenge the animals were caged with male mice and monitored for pregnancy. Mice vaccinated with MOMP+Montanide+CpG developed high levels of C. muridarum-specific antibodies, with a high IgG2a/ IgG1 ratio and neutralizing titres. Animals immunized using Alum+CpG had low antibody levels. Cellular immune responses were significantly higher in mice vaccinated with MOMP and Montanide+CpG, but not with Alum+CpG, when compared with negative controls. In conclusion, this is the first time that a subunit vaccine has been shown to elicit a protective immune response in the highly susceptible C3H/HeN strain of mice against an upper genital challenge.

Implementation of a vaccine is likely the best approach to curtail Chlamydia trachomatis infections. The aim of this study was to determine the ability of a vaccine formulated with the recombinant major outer membrane protein (MOMP) and Th1 and Th2 adjuvants, delivered by combinations of systemic and mucosal routes, to elicit long-term protection in mice against a genital challenge with Chlamydia muridarum. As a negative control, mice were vaccinated with the recombinant Neisseria gonorrhoeae porinB, and the positive control group was immunized with C. muridarum live elementary bodies (EB). The four vaccines formulated with MOMP, as determined by the titers of IgG and neutralizing antibodies in serum, proliferative responses of T-cells stimulated with EB and levels of IFN-γ in the supernatants, elicited robust humoral and cellular immune responses over a 6-month period. Groups of mice were challenged genitally at 60, 120, or 180 days postimmunization. Based on the number of mice with positive vaginal cultures, number of positive cultures, length of time of shedding, and number of inclusion forming units recovered, MOMP vaccinated groups were significantly protected. To assess fertility, when the vaginal cultures became negative, female mice were caged with male mice and the outcome of the pregnancy evaluated. As determined by the number of pregnant mice and the number of embryos, two of the vaccine formulations protected mice up to 180 days postimmunization. To our knowledge this is the first subunit of Chlamydia vaccine that has elicited in mice significant long-term protection against a genital challenge.