In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Risk factors for lymphatic filariasis and mass drug administration non-participation in Mandalay Region, Myanmar.

BACKGROUND: Myanmar commenced a lymphatic filariasis (LF) elimination programme in 2000. Whilst the country has made considerable progress since then, a number of districts have demonstrated persistent transmission after many rounds of mass drug administration (MDA). The causes of unsuccessful MDA have been examined elsewhere; however, there remains little information on the factors that contribute in Myanmar. METHODS: We conducted an analysis of factors associated with persistent infection, LF-related hydrocoele and MDA participation in an area with ongoing transmission in 2015. A cross-sectional household survey was undertaken in 24 villages across four townships of Mandalay Region. Participants were screened for circulating filarial antigen (CFA) using immunochromatographic tests and, if positive, for microfilaria by night-time thick blood slide. Individuals 15 year and older were assessed for filariasis morbidity (lymphoedema and, if male, hydrocoele) by ultrasound-assisted clinical examination. A pre-coded questionnaire was used to assess risk factors for LF and for non-participation (never taking MDA). Significant variables identified in univariate analyses were included in separate step-wise multivariate logistic regressions for each outcome. RESULTS: After adjustment for covariates and survey design, being CFA positive was significantly associated with age [odds ratio (OR) 1.03, 95% CI 1.01-1.06), per year], male gender (OR 3.14, 1.27-7.76), elevation (OR 0.96, 0.94-0.99, per metre) and the density of people per household room (OR 1.59, 1.31-1.92). LF-related hydrocoele was associated with age (OR 1.06, 1.03-1.09, per year) and residing in Amarapura Township (OR 8.93, 1.37-58.32). Never taking MDA was associated with male gender [OR 6.89 (2.13-22.28)] and age, particularly in females, with a significant interaction term. Overall, compared to those aged 30-44 years, the proportion never taking MDA was higher in all age groups (OR highest in those < 5 years and > 60 years, ranging from 3.37 to 12.82). Never taking MDA was also associated with residing in Amarapura township (OR 2.48, 1.15-5.31), moving to one's current village from another (OR 2.62, 1.12-6.11) and ever having declined medication (OR 11.82, 4.25-32.91). Decreased likelihood of never taking MDA was associated with a higher proportion of household members being present during the last MDA round (OR 0.16, 0.03-0.74) and the number visits by the MDA programme (OR 0.69, 0.48-1.00). CONCLUSIONS: These results contribute to the understanding of LF and MDA participation-related risk factors and will assist Myanmar to improve its elimination and morbidity management programmes.

Preventive chemotherapy reverses covert, lymphatic-associated tissue change in young people with lymphatic filariasis in Myanmar.

OBJECTIVES: This longitudinal comparative study investigated the effect of preventive chemotherapy (PC) on covert tissue changes associated with lymphatic filariasis (LF) among young people living in an LF-endemic area in Myanmar. METHODS: Tissue compressibility and extracellular free fluid in the lower limbs of people aged 10-21 years were measured using indurometry and bioimpedance spectroscopy (BIS). Baseline measures were taken in October 2014, annual mass drug administration (MDA) of PC was delivered in December, and in March 2015 further PC was offered to LF-positive cases who had missed MDA. Follow-up measures were taken in February and June 2015. RESULTS: A total of 50 antigen-positive cases and 46 antigen-negative controls were included. Self-reported PC consumption was 60.1% during 2014 MDA and 66.2% overall. At second follow-up, 24 of 34 cases and 27 of 43 controls had consumed PC. Significant and clinically relevant between-group differences at baseline were not found post-PC. Bayesian linear mixed models showed a significant change in indurometer scores at both calves for antigen-positive cases who consumed any PC (dominant calf: -0.30 [95% CI -0.52, -0.07], P < 0.05 and non-dominant calf: -0.35 [95% CI -0.58, -0.12], P < 0.01). Changes in antigen-negative participants or those not consuming PC were not significant. CONCLUSION: This study is the first attempt to use simple field-friendly tools to track fluid and tissue changes after treatment of asymptomatic people infected with LF. Results suggested that PC alone is sufficient to reverse covert lymphatic disturbance. Longer follow-up of larger cohorts is required to confirm these improvements and whether they persist over time. These findings should prompt increased efforts to overcome low PC coverage, which misses many infected young people, particularly males, who are unaware of their infection status, unmotivated to take PC and at risk of developing lymphoedema. Indurometry and BIS should be considered in assessment of lymphatic filariasis-related lymphedema.

OBJECTIFS: Cette étude comparative longitudinale a investigué l'effet de la chimiothérapie préventive (CP) sur les modifications tissulaires cachées associées à la filariose lymphatique (FL) chez les jeunes vivant dans une zone d'endémie pour la FL au Myanmar. MÉTHODES: La compressibilité des tissus et le liquide libre extracellulaire dans les membres inférieurs des personnes âgées de 10 à 21 ans ont été mesurés par indurométrie et spectroscopie de bioimpédance (BIS). Les mesures de base ont été prises en octobre 2014, la distribution en masse de médicament (DMM) annuelle a été administrée en décembre et en mars 2015, et une CP additionnelle a été offerte aux cas positifs pour la FL qui avaient manqué la DMM. Des mesures de suivi ont été prises en février et juin 2015. RÉSULTATS: 50 cas positifs pour l'antigène et 46 témoins négatifs ont été inclus. L'administration de CP auto-déclarée était de 60,1% durant la DMM de 2014 et de 66,2% au total. Au deuxième suivi, 24 des 34 cas et 27 des 43 témoins avaient pris la CP. Des différences significatives et cliniquement pertinentes entre les groupes au départ n'ont pas été trouvées après la CP. Les modèles mixtes linéaires bayésiens ont montré un changement significatif des scores d'indurometrie aux deux mollets pour les cas positifs pour l'antigène qui prenaient une CP (mollet dominant: -0,30 [IC95%: -0,52, -0,07], p <0,05, mollet non dominant: - 0,35 [IC95%: -0,58, -0,12], p <0,01). Les changements chez les participants négatifs pour l'antigène ou ceux qui ne prenaient pas de CP n'étaient pas significatifs. CONCLUSION: Cette étude est la première tentative d'utilisation d'outils simples, conviviaux sur le terrain, pour suivre les modifications du tissu conjonctif après le traitement de personnes asymptomatiques infectées par la FL. Les résultats suggèrent que la CP seule est suffisante pour inverser les modifications lymphatiques cachées. Un suivi plus long de plus grandes cohortes est nécessaire pour confirmer ces améliorations et déterminer si elles persistent ou non. Ces résultats devraient inciter à redoubler d'efforts pour surmonter la faible couverture en CP, qui rate beaucoup de jeunes infectés, en particulier les hommes, qui ne sont pas au courant de leur statut d'infection, qui ne sont pas motivés pour prendre une CP et risquent de développer un lymphœdème. L'indurométrie et la BIS devraient être considérées dans l'évaluation du lymphoedème associé à la filariose lymphatique.

The prevalence of lymphatic filariasis infection and disease following six rounds of mass drug administration in Mandalay Region, Myanmar.

Lymphatic filariasis is widely endemic in Myanmar. Despite the establishment of an elimination program in 2000, knowledge of the remaining burden of disease relies predominantly on programmatic information. To assist the program, we conducted an independent cross-sectional household cluster survey to determine the prevalence of filariasis infection, morbidity and mass-drug administration coverage in four townships of the Mandalay Region: Amarapura, Patheingyi, Tada-U and Wundwin. The survey included 1014 individuals from 430 randomly selected households in 24 villages. Household members one year and older were assessed for antigenaemia using immunochromatographic test cards and if positive, microfilaraemia by night-time thick blood smear. Participants 15 years and older were assessed for filariasis morbidity by ultrasound-assisted clinical examination. The overall prevalence of infection was 2.63% by antigenaemia (95% confidence interval (CI) 1.71-4.04%) and 1.03% by microfilaraemia (95%CI 0.59-1.47%). The prevalence of hydrocoele in adult males was 2.78% (95%CI 1.23-6.15%) and of lymphoedema in both genders was 0% (95%CI 0-0.45%). These results indicate the persistence of filarial infection and transmission despite six rounds of annual mass drug administration and highlight the need for further rounds as well as the implementation of morbidity management programs in the country.

Protection against inhalation anthrax by immunization with Salmonella enterica serovar Typhi Ty21a stably producing protective antigen of Bacillus anthracis.

The national blueprint for biodefense concluded that the United States is underprepared for biological threats. The licensed anthrax vaccine absorbed vaccine, BioThrax, requires administration of at least 3-5 intramuscular doses. The anthrax vaccine absorbed vaccine consists of complex cell-free culture filtrates of a toxigenic Bacillus anthracis strain and causes tenderness at the injection site and significant adverse events. We integrated a codon-optimized, protective antigen gene of B. anthracis (plus extracellular secretion machinery), into the chromosome of the licensed, oral, live-attenuated typhoid fever vaccineTy21a to form Ty21a-PA-01 and demonstrated excellent expression of the gene encoding protective antigen. We produced the vaccine in a 10-L fermenter; foam-dried and vialed it, and characterized the dried product. The vaccine retained ~50% viability for 20 months at ambient temperature. Sera from animals immunized by the intraperitoneal route had high levels of anti-protective antigen antibodies by enzyme-linked immunosorbent assay and anthrax lethal toxin-neutralizing activity. Immunized mice were fully protected against intranasal challenge with ~5 LD50 of B. anthracis Sterne spores, and 70% (7/10) of vaccinated rabbits were protected against aerosol challenge with 200 LD50 of B. anthracis Ames spores. There was a significant correlation between protection and antibody levels determined by enzyme-linked immunosorbent assay and toxin-neutralizing activity. These data provide the foundation for achievement of our ultimate goal, which is to develop an oral anthrax vaccine that is stable at ambient temperatures and induces the rapid onset of durable, high-level protection after a 1-week immunization regimen.

Development of a Live Attenuated Bivalent Oral Vaccine Against Shigella sonnei Shigellosis and Typhoid Fever.

Shigella sonnei and Salmonella Typhi cause significant morbidity and mortality. We exploited the safety record of the oral, attenuated S. Typhi vaccine (Ty21a) by using it as a vector to develop a bivalent oral vaccine to protect against S. sonnei shigellosis and typhoid fever. We recombineered the S. sonnei form I O-antigen gene cluster into the Ty21a chromosome to create Ty21a-Ss, which stably expresses S. sonnei form I O antigen. To enhance survivability in the acid environment of the stomach, we created an acid-resistant strain, Ty21a-AR-Ss, by inserting Shigella glutaminase-glutamate decarboxylase systems coexpressed with S. sonnei form I O-antigen gene. Mice immunized intranasally with Ty21a-AR-Ss produced antibodies against S. sonnei and S. Typhi, and survived lethal intranasal S. sonnei challenge. This paves the way for proposed good manufacturing practices manufacture and clinical trials intended to test the clinical effectiveness of Ty21a-AR-Ss in protecting against S. sonnei shigellosis and typhoid fever, as compared with the current Ty21a vaccine.

Lymphatic Filariasis Increases Tissue Compressibility and Extracellular Fluid in Lower Limbs of Asymptomatic Young People in Central Myanmar.

When normal lymphatic function is hampered, imperceptible subcutaneous edema can develop and progress to overt lymphedema. Low-cost reliable devices for objective assessment of lymphedema are well accepted in clinical practice and research on breast-cancer related lymphedema but are untested in populations with lymphatic filariasis (LF). This is a cross-sectional analysis of baseline data in a longitudinal study on asymptomatic, LF antigen-positive and -negative young people in Myanmar. Rapid field screening was used to identify antigen-positive cases and a group of antigen-negative controls of similar age and gender were invited to continue in the study. Tissue compressibility was assessed with three tissue tonometers, and free fluids were assessed using bio-impedance spectroscopy (BIS). Infection status was confirmed by Og4C3 antigen assay. At baseline (n = 98), antigen-positive cases had clinically relevant increases in tissue compressibility at the calf using a digital Indurometer (11.1%, p = 0.021), and in whole-leg free fluid using BIS (9.2%, p = 0.053). Regression analysis for moderating factors (age, gender, hydration) reinforced the between-infection group differences. Results demonstrate that sub-clinical changes associated with infection can be detected in asymptomatic cases. Further exploration of these low-cost devices in clinical and research settings on filariasis-related lymphedema are warranted.

Genome Sequence of Salmonella enterica Serovar Typhi Oral Vaccine Strain Ty21a.

Attenuated Salmonella enterica serovar Typhi strain Ty21a is an important vaccine for controlling typhoid fever and serves as an oral vector for delivering heterologous antigens. The key attenuating features of this randomly mutated strain remain in question. Genome sequencing has revealed 679 single nucleotide polymorphisms (SNPs), and will help define alterations contributing to Ty21a safety and immunogenicity.

Stable expression of Shigella sonnei form I O-polysaccharide genes recombineered into the chromosome of live Salmonella oral vaccine vector Ty21a.

Live, attenuated Salmonella enterica serovar Typhi strain Ty21a, a licensed oral typhoid fever vaccine, has also been employed for use as a vector to deliver protective antigens of Shigella and other pathogens. Importantly, lipopolysaccharide (LPS) alone has been shown to be a potent antigen for specific protection against shigellosis. We reported previously the plasmid cloning of heterologous LPS biosynthetic genes and the expression in Ty21a of either S. sonnei or of S. dysenteriae 1 LPS's. The resulting plasmids encoding Shigella LPS's were reasonably stable for >50 generations of growth in nonselective media, but still contained an antibiotic resistance marker that is objectionable to vaccine regulatory authorities. Deletion of this antibiotic-resistance marker inexplicably resulted in significant plasmid instability. Thus, we sought a method to insert the large ∼12kb S. sonnei LPS gene region into the chromosome, that would allow for subsequent removal of a selectable marker and would result in 100% genetic stability. Toward this objective, we optimized an existing recombination method to mediate the insertion of a ∼12kb region encoding the S. sonnei LPS genes into the Ty21a genome in a region that is nonfunctional due to mutation. The resulting strain Ty21a-Ss simultaneously expresses both homologous Ty21a and heterologous S. sonnei O-antigens. This chromosomal insert was shown to be 100% genetically stable in vitro and in vivo. Moreover, Ty21a-Ss elicited strong dual anti-LPS serum immune responses and 100% protection in mice against a virulent S. sonnei challenge. This new vaccine candidate, absolutely stable for vaccine manufacture, should provide combined protection against enteric fevers due to Salmonella serovar Typhi as shown previously (and some Paratyphi infections) and against shigellosis due to S. sonnei.

Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: preclinical evidence of immunogenicity and protection.

Shigella dysenteriae serotype 1 (S. dysenteriae 1) causes severe shigellosis that is typically associated with high mortality. Antibodies against Shigella serotype-specific O-polysaccharide (O-Ps) have been shown to be host protective. In this study, the rfb locus and the rfp gene with their cognate promoter regions were PCR-amplified from S. dysenteriae 1, cloned, and sequenced. Deletion analysis showed that eight rfb ORFs plus rfp are necessary for biosynthesis of this O-Ps. A tandemly-linked rfb-rfp gene cassette was cloned into low copy plasmid pGB2 to create pSd1. Avirulent Salmonella enterica serovar Typhi (S. Typhi) Ty21a harboring pSd1 synthesized S. Typhi 9, 12 LPS as well as typical core-linked S. dysenteriae 1 LPS. Animal immunization studies showed that Ty21a (pSd1) induces protective immunity against high stringency challenge with virulent S. dysenteriae 1 strain 1617. These data further demonstrate the utility of S. Typhi Ty21a as a live, bacterial vaccine delivery system for heterologous O-antigens, supporting the promise of a bifunctional oral vaccine for prevention of shigellosis and typhoid fever.