Genetic diversity of G9, G3, G8 and G1 rotavirus group A strains circulating among children with acute gastroenteritis in Vietnam from 2016 to 2021.

Rotavirus group A (RVA) is the most common cause of severe childhood diarrhea worldwide. The introduction of rotavirus vaccination programs has contributed to a reduction in hospitalizations and mortality caused by RVA. From 2016 to 2021, we conducted surveillance to monitor RVA prevalence and genotype distribution in Nam Dinh and Thua Thien Hue (TT Hue) provinces where a pilot Rotavin-M1 vaccine (Vietnam) implementation took place from 2017 to 2020. Out of 6626 stool samples, RVA was detected in 2164 (32.6%) by ELISA. RT-PCR using type-specific primers were used to determine the G and P genotypes of RVA-positive specimens. Whole genome sequences of a subset of 52 specimens randomly selected from 2016 to 2021 were mapped using next-generation sequencing. From 2016 to 2021, the G9, G3 and G8 strains dominated, with detected frequencies of 39%, 23%, and 19%, respectively; of which, the most common genotypes identified were G9P[8], G3P[8] and G8P[8]. G1 strains re-emerged in Nam Dinh and TT Hue (29.5% and 11.9%, respectively) from 2020 to 2021. G3 prevalence decreased from 74% to 20% in TT Hue and from 21% to 13% in Nam Dinh province between 2017 and 2021. The G3 strains consisted of 52% human typical G3 (hG3) and 47% equine-like G3 (eG3). Full genome analysis showed substantial diversity among the circulating G3 strains with different backgrounds relating to equine and feline viruses. G9 prevalence decreased sharply from 2016 to 2021 in both provinces. G8 strains peaked during 2019-2020 in Nam Dinh and TT Hue provinces (68% and 46%, respectively). Most G8 and G9 strains had no genetic differences over the surveillance period with very high nucleotide similarities of 99.2-99.9% and 99.1-99.7%, respectively. The G1 strains were not derived from the RVA vaccine. Changes in the genotype distribution and substantial diversity among circulating strains were detected throughout the surveillance period and differed between the two provinces. Determining vaccine effectiveness against circulating strains over time will be important to ensure that observed changes are due to natural secular variation and not from vaccine pressure.

Development and Qualification of an Antigen Integrity Assay for a Plasmodium falciparum Malaria Transmission Blocking Vaccine Candidate, Pfs230.

During development of a subunit vaccine, monitoring integrity of the recombinant protein for process development and quality control is critical. Pfs230 is a leading malaria transmission blocking vaccine candidate and the first to reach a Phase 2 clinical trial. The Pfs230 protein is expressed on the surface of gametes, and plays an important role in male fertility. While the potency of Pfs230 protein can be determined by a standard membrane-feeding assay (SMFA) using antibodies from immunized subjects, the precision of a general in vivo potency study is known to be poor and is also time-consuming. Therefore, using a well-characterized Pfs230 recombinant protein and two human anti-Pfs230 monoclonal antibodies (mAbs), which have functional activity judged by SMFA, a sandwich ELISA-based in vitro potency assay, called the Antigen Integrity Assay (AIA), was developed. Multiple validation parameters of AIA were evaluated to qualify the assay following International Conference on Harmonization (ICH) Q2(R1) guidelines. The AIA is a high throughput assay and demonstrated excellent precision (3.2 and 5.4% coefficients of variance for intra- and inter-assay variability, respectively) and high sensitivity (>12% impurity in a sample can be detected). General methodologies and the approach to assay validation described herein are amenable to any subunit vaccine as long as more than two functional, non-competing mAbs are available. Thus, this study supports future subunit vaccine development.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses.

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1-2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

Elucidating functional epitopes within the N-terminal region of malaria transmission blocking vaccine antigen Pfs230.

Pfs230 is a leading malaria transmission blocking vaccine (TBV) candidate. Comprising 3135 amino acids (aa), the large size of Pfs230 necessitates the use of sub-fragments as vaccine immunogens. Therefore, determination of which regions induce functional antibody responses is essential. We previously reported that of 27 sub-fragments spanning the entire molecule, only five induced functional antibodies. A "functional" antibody is defined herein as one that inhibits Plasmodium falciparum parasite development in mosquitoes in a standard membrane-feeding assay (SMFA). These five sub-fragments were found within the aa 443-1274 range, and all contained aa 543-730. Here, we further pinpoint the location of epitopes within Pfs230 that are recognized by functional antibodies using antibody depletion and enrichment techniques. Functional epitopes were not found within the aa 918-1274 region. Within aa 443-917, further analysis showed the existence of functional epitopes not only within the aa 543-730 region but also outside of it. Affinity-purified antibodies using a synthetic peptide matching aa 543-588 showed activity in the SMFA. Immunization with a synthetic peptide comprising this segment, formulated either as a carrier-protein conjugate vaccine or with a liposomal vaccine adjuvant system, induced antibodies in mice that were functional in the SMFA. These findings provide key insights for Pfs230-based vaccine design and establish the feasibility for the use of synthetic peptide antigens for a malaria TBV.

Poor CD4+ T Cell Immunogenicity Limits Humoral Immunity to P. falciparum Transmission-Blocking Candidate Pfs25 in Humans.

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4+ and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4+ T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4+/Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission.

Strong concordance between percent inhibition in oocyst and sporozoite intensities in a Plasmodium falciparum standard membrane-feeding assay.

BACKGROUND: Effective malaria transmission-blocking vaccines (TBVs) can support malaria eradication programmes, and the standard membrane-feeding assay (SMFA) has been used as a "gold standard" assay for TBV development. However, in SMFA, the inhibitory activity is commonly measured at oocyst stage of parasites, while it is the sporozoites which transmit malaria from a mosquito to a human. A handful of studies have shown that there is a positive correlation between oocyst and sporozoite intensities. However, no study has been completed to compare inhibition levels in oocyst and sporozoite intensities in the presence of transmission-blocking (TB) antibodies. RESULTS: Plasmodium falciparum NF54 gametocytes were fed to Anopheles stephensi mosquitoes with or without anti-Pfs25 or anti-Pfs48/45 TB antibodies in 15 independent assays. For each group, a portion of the mosquitoes was dissected for oocyst counts (day 8 after feed), and a portion of the remaining mosquitoes was dissected for sporozoite counts (day 16). This study covered a large range of oocyst and sporozoite intensities: 0.2 to 80.5 on average for oocysts, and 141 to 77,417 for sporozoites. The sporozoite data were well explained by a zero-inflated negative binomial model, regardless of the presence or absence of TB antibodies. Inhibition levels in both oocyst and sporozoite intensities were determined within the same groups in 9 independent assays. When the level of inhibition in sporozoite number (expressed as Log Mean Ratio, LMR; average number in a control group was divided by the one in a test group, then took a log of the ratio) was plotted against LMR in oocyst number, the best-fit slope of a linear regression was not different from 1 (the best estimate, 1.08; 95% confidence interval, 0.87 to 1.29). Furthermore, a Bland-Altman analysis showed a strong agreement between inhibitions in oocysts and in sporozoites. CONCLUSIONS: The results indicate that percent inhibition in oocyst intensity of a test sample can be directly converted to % inhibition in sporozoite intensity in P. falciparum SMFA. Therefore, if sporozoite intensity determines transmission rate from mosquitoes to humans, the percent inhibition in oocyst intensity measured by SMFA can be used to estimate the TBV efficacy.

ELISA units, IgG subclass ratio and avidity determined functional activity of mouse anti-Pfs230 antibodies judged by a standard membrane-feeding assay with Plasmodium falciparum.

The standard membrane-feeding assay (SMFA) is a functional assay that has been used to inform the development of transmission-blocking vaccines (TBV) against Plasmodium falciparum malaria. For Pfs230, a lead target antigen for TBV development, a few studies have tested either a single anti-Pfs230 polyclonal or monoclonal antibody (one antibody per study) at serial dilutions and showed a dose-dependent response. Further, there have been reports that the SMFA activity of anti-Pfs230 polyclonal and monoclonal antibodies were enhanced in the presence of complement. However, no analysis has been performed with multiple samples, and the impact of anti-Pfs230 antibody titers, IgG subclass profile and avidity were evaluated together in relation to transmission-reducing activity (TRA) by SMFA. In this report, a total of 39 unique anti-Pfs230 IgGs from five different mouse immunization studies were assessed for their ELISA units (EU), IgG2/IgG1 ratio and avidity by ELISA, and the functionality (% transmission-reducing activity, %TRA) by SMFA. The mice were immunized with Pfs230 alone, Pfs230 conjugated to CRM197, or a mixture of unconjugated Pfs230 and CRM197 proteins using Alhydrogel or Montanide ISA720 adjuvants. In all studies, the Pfs230 antigen was from the same source. There was a significant correlation between EU and %TRA (p < 0.0001 by a Spearman rank test) for the anti-Pfs230 IgGs. Notably, multiple linear regression analyses showed that both IgG2/IgG1 ratio and avidity significantly affected %TRA (p = 0.003 to p = 0.014, depending on the models) after adjusting for EU. The results suggest that in addition to antibody titers, IgG2/IgG1 ratio and avidity should each be evaluated to predict the biological activity of anti-Pfs230 antibodies for future vaccine development.

Malaria Derived Glycosylphosphatidylinositol Anchor Enhances Anti-Pfs25 Functional Antibodies That Block Malaria Transmission.

Malaria, one of the most common vector borne human diseases, is a major world health issue. In 2015 alone, more than 200 million people were infected with malaria, out of which, 429 000 died. Even though artemisinin-based combination therapies (ACT) are highly effective at treating malaria infections, novel efforts toward development of vaccines to prevent transmission are still needed. Pfs25, a postfertilization stage parasite surface antigen, is a leading transmission-blocking vaccine (TBV) candidate. It is postulated that Pfs25 anchors to the cell membrane using a glycosylphosphatidylinositol (GPI) linker, which itself possesses pro-inflammatory properties. In this study, Escherichia coli derived extract (XtractCF+TM) was used in cell free protein synthesis [CFPS] to successfully express >200 mg/L of recombinant Pfs25 with a C-terminal non-natural amino acid (nnAA), namely, p-azidomethyl phenylalanine (pAMF), which possesses a reactive azide group. Thereafter, a unique conjugate vaccine (CV), namely, Pfs25-GPI was generated with dibenzocyclooctyne (DBCO) derivatized glycan core of malaria GPI using a simple but highly efficient copper free click chemistry reaction. In mice immunized with Pfs25 or Pfs25-GPI, the Pfs25-GPI group showed significantly higher titers compared to the Pfs25 group. Moreover, only purified IgGs from Pfs25-GPI group were able to significantly block transmission of parasites to mosquitoes, as judged by a standard membrane feeding assay [SMFA]. To our knowledge, this is the first report of the generation of a CV using Pfs25 and malaria specific GPI where the GPI is shown to enhance the ability of Pfs25 to elicit transmission blocking antibodies.

Transmission-blocking activity is determined by transmission-reducing activity and number of control oocysts in Plasmodium falciparum standard membrane-feeding assay.

Malaria transmission-blocking vaccines (TBVs) are potentially helpful tools for malaria eradication. The standard membrane-feeding assay (SMFA) is considered one of the "gold standard" assays for TBV development. However, lack of consensus in reporting results from SMFA has made it very challenging to compare results from different studies. Two main readouts, % inhibition in mean oocyst count per mosquito (TRA) and % inhibition in prevalence of infected mosquitoes (TBA), have been used widely. In this study, we statistically modeled the oocyst data in SMFA using data from 105 independent feeding experiments including 9804 mosquitoes. The model was validated using an independent data set that included 10,790 mosquitoes from 110 feeding studies. The model delineates a relationship between TRA, the mean oocyst count in the control mosquitoes (mo-contl), and TBA. While TRA was independent from mo-contl, TBA values changed depending on mo-contl. Regardless of monoclonal or polyclonal antibodies tested, there were strong concordances between observed TBA and predicted TBA based on the model using mo-contl and observed TRA. Simulations showed that SMFA with lower true control means had increased uncertainty in TRA estimates. The strong linkage between TBA, TRA and mo-contl inspired creation of a standardized TBA, a model-based TBA standardized to a target control mean, which allows comparison across multiple feeds regardless of mo-contl. This is the first study showing that the observed TBA can be reasonably predicted by mo-contl and the TRA of the test antibody using independent experimental data. This study indicates that TRA should be used to compare results from multiple feeds with different levels of mo-contl. If a measure of TBA is desired, it is better to report standardized TBA rather than observed TBA. These recommendations support rational comparisons of results from different studies, thus benefiting future TBV development.