Innovative method for rapid detection of falsified COVID-19 vaccines through unopened vials using handheld Spatially Offset Raman Spectroscopy (SORS)

Preventing, detecting, and responding to substandard and falsified vaccines is of critical importance for ensuring the safety, efficacy, and public trust in vaccines. This is of heightened importance in context of public health crisis, such as the COVID-19 pandemic, in which extreme world-wide shortages of vaccines provided a fertile ground for exploitation by falsifiers. Here, a proof-of-concept study explored the feasibility of using a handheld Spatially Offset Raman Spectroscopy (SORS) device to authenticate COVID-19 vaccines through rapid analysis of unopened vaccine vials. The results show that SORS can verify the chemical identity of dominant excipients non-invasively through vaccine vial walls. The ability of SORS to identify potentially falsified COVID-19 vaccines was demonstrated by measurement of surrogates for falsified vaccines contained in vaccine vials. In all cases studied, the SORS technique was able to differentiate between surrogate samples from the genuine COVISHIELD™ vaccine. The genuine vaccines tested included samples from six batches across two manufacturing sites to account for any potential variations between batches or manufacturing sites. Batch and manufacturing site variations were insignificant. In conjunction with existing security features, for example on labels and packaging, SORS provided an intrinsic molecular fingerprint of the dominant excipients of the vaccines. The technique could be extended to other COVID-19 and non-COVID-19 vaccines, as well as other liquid medicines. As handheld and portable SORS devices are commercially available and widely used for other purposes, such as airport security, they are rapidly deployable non-invasive screening tools for vaccine authentication.</p

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin

BackgroundLuminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT).MethodsThe MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT.ResultsWe identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was &lt; 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was &lt; 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (&gt; 0.75) with toxin neutralization assays for PT and DT was observed.ConclusionThe MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity

Safety and efficacy of malaria vaccine candidate R21/Matrix-M in African children: a multicentre, double-blind, randomised, phase 3 trial

Background Recently, we found that a new malaria vaccine, R21/Matrix-M, had over 75% efficacy against clinical malaria with seasonal administration in a phase 2b trial in Burkina Faso. Here, we report on safety and efficacy of the vaccine in a phase 3 trial enrolling over 4800 children across four countries followed for up to 18 months at seasonal sites and 12 months at standard sites. Methods We did a double-blind, randomised, phase 3 trial of the R21/Matrix-M malaria vaccine across five sites in four African countries with differing malaria transmission intensities and seasonality. Children (aged 5–36 months) were enrolled and randomly assigned (2:1) to receive 5 μg R21 plus 50 μg Matrix-M or a control vaccine (licensed rabies vaccine [Abhayrab]). Participants, their families, investigators, laboratory teams, and the local study team were masked to treatment. Vaccines were administered as three doses, 4 weeks apart, with a booster administered 12 months after the third dose. Half of the children were recruited at two sites with seasonal malaria transmission and the remainder at standard sites with perennial malaria transmission using age-based immunisation. The primary objective was protective efficacy of R21/Matrix-M from 14 days after third vaccination to 12 months after completion of the primary series at seasonal and standard sites separately as co-primary endpoints. Vaccine efficacy against multiple malaria episodes and severe malaria, as well as safety and immunogenicity, were also assessed. This trial is registered on ClinicalTrials.gov, NCT04704830, and is ongoing. Findings From April 26, 2021, to Jan 12, 2022, 5477 children consented to be screened, of whom 1705 were randomly assigned to control vaccine and 3434 to R21/Matrix-M; 4878 participants received the first dose of vaccine. 3103 participants in the R21/Matrix-M group and 1541 participants in the control group were included in the modified per-protocol analysis (2412 [51·9%] male and 2232 [48·1%] female). R21/Matrix-M vaccine was well tolerated, with injection site pain (301 [18·6%] of 1615 participants) and fever (754 [46·7%] of 1615 participants) as the most frequent adverse events. Number of adverse events of special interest and serious adverse events did not significantly differ between the vaccine groups. There were no treatment-related deaths. 12-month vaccine efficacy was 75% (95% CI 71–79; p<0·0001) at the seasonal sites and 68% (61–74; p<0·0001) at the standard sites for time to first clinical malaria episode. Similarly, vaccine efficacy against multiple clinical malaria episodes was 75% (71–78; p<0·0001) at the seasonal sites and 67% (59–73; p<0·0001) at standard sites. A modest reduction in vaccine efficacy was observed over the first 12 months of follow-up, of similar size at seasonal and standard sites. A rate reduction of 868 (95% CI 762–974) cases per 1000 children-years at seasonal sites and 296 (231–362) at standard sites occurred over 12 months. Vaccine-induced antibodies against the conserved central Asn-Ala-Asn-Pro (NANP) repeat sequence of circumsporozoite protein correlated with vaccine efficacy. Higher NANP-specific antibody titres were observed in the 5–17 month age group compared with 18–36 month age group, and the younger age group had the highest 12-month vaccine efficacy on time to first clinical malaria episode at seasonal (79% [95% CI 73–84]; p<0·001) and standard (75% [65–83]; p<0·001) sites. Interpretation R21/Matrix-M was well tolerated and offered high efficacy against clinical malaria in African children. This low-cost, high-efficacy vaccine is already licensed by several African countries, and recently received a WHO policy recommendation and prequalification, offering large-scale supply to help reduce the great burden of malaria in sub-Saharan Africa. Funding The Serum Institute of India, the Wellcome Trust, the UK National Institute for Health Research Oxford Biomedical Research Centre, and Open Philanthropy

Evaluation of Critical Quality Attributes of a Pentavalent (A, C, Y, W, X) Meningococcal Conjugate Vaccine for Global Use

Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM197), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine’s critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India

A novel ELISA for quantification of glycoprotein in human rabies vaccines using a clinically proven virus neutralizing human monoclonal antibody

Global efforts on the replacement of the in vivo rabies vaccine potency test (NIH method) with in vitro methods for quantification of immunodominant glycoprotein (GP) in rabies vaccine have made significant progress. We report here, a sandwich ELISA method based on the use of a neutralizing rabies GP site III directed human monoclonal antibody (RAB-1) and a polyclonal GP specific antibody recognizing the intact form of viral GP. The method was shown to be robust, specific, linear, precise and accurate in the quantification of intact GP in vaccine samples. The assay was able to differentiate between potent and sub-potent vaccine samples. The assay was shown to be linear over the range of 0.07–2.25 IU/mL with LOD and LLOQ values of 0.035 and 0.070 IU/mL, respectively. The assay was able to quantify the GP content of rabies vaccines derived from rabies vaccine strains, e.g., Pittman-Moore, Pasteur and Flury LEP with acceptable precision (CV < 20%) and also showed good agreement with NIH potency estimates. The binding kinetics of RAB-1 with intact and modified vaccine samples were also characterized using biolayer interferometry (BLI). The developed method may be used as an alternative to the NIH method in quality control testing of human rabies vaccines

Evaluation of the Impact of Herbicidal Weed Management Practices on Weed Density, Yield Attributes and Yield of Irrigated Chickpea (Cicer arietinum L.)

In the Rabi season of 2021-22, an experiment was conducted at the Agronomy Research Farm of CCS Haryana Agricultural University in Hisar. The aim was to examine how herbicidal weed management affects irrigated chickpea. The experiment was laid out in Randomised Block Design (RBD) with thirteen treatments, each repeated three times. The treatments included various herbicides applied at different stages, such as pre-plant incorporation (PPI), pre-emergence (PRE), and post-emergence (POE). Interestingly, the Ready mix (RM) herbicide application of pendimethalin + imazethapyr (RM) @ 1000 g a.i ha-1, applied both as PPI and PRE, outperformed the herbicides applied solely as PPI, PRE, or POE. Among the herbicidal treatments, the combined during PPI and PRE stages exhibited excellent control over a diverse weed population, leading to a significant increase in chickpea yield compared to the weedy check. The number of seeds per pod, pods per plant, and branches per plant varied significantly with different weed control treatments. Weed-free plots showed the highest values in these parameters. The uncontrolled growth of weeds in the weedy check resulted in a 55.2% reduction in seed yield as compared to weed-free plots. The maximum seed yield (1968 kg ha-1) and favorable yield attributes were observed in the weed- free treatment, statistically comparable to the yield obtained from two hand weeding at 30 and 50 days after sowing (1940 kg ha-1). Among herbicidal treatments, the highest seed yield was achieved with the PRE application of pendimethalin + imazethapyr (RM) @ 1000 g a.i. ha-1 (1827 kg ha-1). The dominant weed flora consisted of Chenopodium album, Fumaria parviflora and Anagallis arvensis. Density of different weed species was significantly influenced by different weed control treatments. All the weed control treatments significantly reduced the total weed density and dry matter accumulation by weeds in comparison to weedy check. Weed free and two hand hoeing reduced the weed population drastically which was statistically at par with PRE application of pendimethalin + imazethapyr (RM) at 1000 g a.i. ha-1. Chenopodium album, Fumaria parviflora and Anagallis arvensis were effectively controlled by RM irrespective of its time of application

Studies on the Effect of Chemical Weed Management on Growth Indices, Yield and Quality of Irrigated Chickpea (Cicer arietinum L.)

Chickpea, characterized by its short stature and slow initial growth, is highly susceptible to a wide range of weed species. The field experiment was designed during the Rabi season of 2021-22 at Agronomy Research Farm, Chaudhary Charan Singh Haryana Agricultural University, Hisar to evaluate the chemical weed management in irrigated chickpea. The experiment was laid out in a Randomised Block Design (RBD) with thirteen treatments replicated thrice. The treatments combinations areT1 (Pendimethalin 30EC @ 1000g a.i ha-1 as PPI), T2 (Imazethapyr 10EC&nbsp; @ 75g a.i ha-1 as PPI), T3 (Imazethapyr 10EC @ 100g a.i ha-1 as PPI), T4 (Pendimethalin 30EC + imazethapyr 2EC (RM) @ 1000g a.i ha-1 as PPI), T5 (Pendimethalin 30EC @ 1000g a.i ha-1 as PRE), T6 (Imazethapyr 10EC @ 75g a.i ha-1 as PRE), T7 (Imazethapyr 10EC @ 100g a.i ha-1 as PRE),&nbsp; T8 (Pendimethalin 30EC&nbsp; + Imazethapyr 2EC (RM) @ 1000g a.i ha-1 as PRE), T9 (Imazethapyr 10EC @ 75g a.i ha-1 as POE), T10 (Imazethapyr 10EC @ 100g a.i ha-1 as POE), T11 (Two hand hoeing at 30 &amp; 50 DAS), T12 (weed free) and T13 (weedy check). The results of this study showed that PPI and PRE application of any herbicide did not cause any phytotoxic effect on chickpea. The Ready mix (RM) herbicide applied as PPI and PRE performed better than sole PPI, PRE, and POE herbicides. Among herbicidal treatments, PPI and PRE application of pendimethalin + imazethapyr (RM) @ 1000 g a.i ha-1 gave excellent control of complex weed flora and increased the yield of chickpea significantly over the weedy check. Different weed control treatments could not bring significant variation in plant population in the initial stage but the plant population declined at maturity. The maximum plant growth attributes were observed with the application of pendimethalin + imazethapyr (RM) @ 1000 g a.i ha-1 as PRE. The POE application of imazethapyr irrespective of the dose recorded the lowest dry matter accumulation throughout the growing season of chickpea. The unchecked growth of weeds in the weedy check caused a 55.2% reduction in seed yield as compared to weed-free treatment. The maximum seed yield (1968 kg ha-1) and yield attributes of chickpea were recorded with weed-free treatment and among herbicidal treatments, maximum seed yield (1827 kg ha-1) was recorded with application of pendimethalin + imazethapyr (RM) @ 1000 g ha-1 PRE

Characterization of Bordetella pertussis Strains Isolated from India

Despite high level vaccination and the availability of two different types of vaccines, whole cell (wP) and acellular vaccines (aP), the resurgence of pertussis has been reported in many countries. Antigenic variation within circulating and vaccine strains is the most documented reason reported for the resurgence of pertussis. Research on genetic divergence among circulating and vaccine strains has largely been reported in countries using aP vaccines. There are inadequate data available for antigenic variation in B. pertussis from wP-using countries. India has used wP for more than 40 years in their primary immunization program. The present study reports five clinical isolates of B. pertussis from samples of pediatric patients with pertussis symptoms observed in India. Genotypic and phenotypic characterization of clinical isolates were performed by serotyping, genotyping, whole genome analyses and comparative genomics. All clinical isolates showed serotype 1, 2 and 3 based on the presence of fimbriae 2 and 3. Genotyping showed genetic similarities in allele types for five aP genes within vaccine strains and clinical isolates reported from India. The presence of the ptxP3 genotype was observed in two out of five clinical isolates. Whole-genome sequencing was performed for clinical isolates using the hybrid strategy of combining Illumina (short reads) and oxford nanopore (long reads) sequencing strategies. Clinical isolates (n = 5) and vaccine strains (n = 7) genomes of B. pertussis from India were compared with 744 B. pertussis closed genomes available in the public databases. The phylogenomic comparison of B. pertussis genomes reported from India will be advantageous in better understanding pertussis resurgence reported globally with respect to pathogen adaptation

Combined Diagnostic Accuracy of Diffusion and Perfusion MR Imaging to Differentiate Radiation-Induced Necrosis from Recurrence in Glioblastoma

We aimed to use quantitative values derived from perfusion and diffusion-weighted MR imaging (PWI and DWI) to differentiate radiation-induced necrosis (RIN) from tumor recurrence in Glioblastoma (GBM) and investigate the best parameters for improved diagnostic accuracy and clinical decision-making. Methods: A retrospective analysis of follow-up MRI with new enhancing observations was performed in histopathologically confirmed subjects of post-treated GBM, who underwent re-surgical exploration. Quantitative estimation of rCBV (relative cerebral blood volume) from PWI and three methods of apparent diffusion coefficient (ADC) estimation were performed, namely ADC R1 (whole cross-sectional area of tumor), ADC R2 (only solid enhancing lesion), and ADC R3 (central necrosis). ROC curve and logistic regression analysis was completed. A confusion matrix table created using Excel provided the best combination parameters to ameliorate false-positive and false-negative results. Results: Forty-four subjects with a mean age of 46 years (range, 19–70 years) underwent re-surgical exploration with RIN in 28 (67%) and recurrent tumor in 16 (33%) on histopathology. rCBV threshold of >3.4 had the best diagnostic accuracy (AUC = 0.93, 81% sensitivity and 89% specificity). A multiple logistic regression model showed significant contributions from rCBV (p p = 0.001). After analysis of confusion matrix ADC R3 > 2032 × 10−6 mm2 achieved 100% specificity with gain in sensitivity (94% vs. 56%). Conclusions: A combination of parameters had better diagnostic performance, and a stepwise combination of rCBV and ADC R3 obviated unnecessary biopsies in 10% (3/28), leading to improved clinical decision-making

A Phase I study to evaluate safety and tolerability of DTaP-IPV + Hib vaccine in healthy adult volunteers in India

Background: To assess safety and tolerability of a diphtheria and tetanus toxoid, acellular pertussis, inactivated poliovirus and Haemophilus influenza type B conjugate adsorbed vaccine (DTaP-IPV + Hib), manufactured by Serum Institute of India Pvt. Ltd. (SIIPL)’s, the current first-in-human Phase 1 study was conducted in healthy adults. Methods: Vaccine was administered as a single 0.5 mL dose intramuscularly into deltoid muscle of 24 healthy adults aged 18–45 years, who were then followed prospectively for one month for safety outcomes. Results: All 24 participants completed the study in compliance with protocol. Four solicited adverse events were reported in three participants during the study; all adverse events were mild and recovered completely. No deaths, unsolicited adverse events, or serious adverse events were reported. Conclusion: SIIPL DTaP-IPV + Hib vaccine was well tolerated and safe in study subjects. Further clinical development will be conducted to assess safety and immunogenicity in young children, the target population.Clinical Trial Registration: CTRI/2017/07/009034