Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214 mg/L in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over four weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80–100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25–250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Field studies were conducted at the Central Potato Research Institute Campus, Modipuram (India), during two seasons (2003-2004 and 2004-2005), with two processing cultivars (cvs Kufri Chipsona-1 and Kufri Chipsona-2) and nine combinations of potassium source (K-chloride, K-sulphate and K-nitrate) and time of K application (basal dressing, split application and split application + foliar spray) to investigate their effect on tuber yield and processing attributes for crisp production under subtropical conditions. Yearly variations were observed for most of the variables studied. Between cultivars, the tuber and biomass yield was higher in cv. Kufri Chipsona-1, whereas specific gravity, tuber dry-matter percentage and crisp recovery were higher in cv. Kufri Chipsona-2. Stem number, plant height and compound leaf number were not influenced by sources of K fertilizer or application time. Processing-grade tuber yield, total tuber yield and biomass yield were statistically similar for K-chloride and K-sulphate and lower for K-nitrate. K sources affected both specific gravity and tuber dry-matter percentage significantly; Ksulphate and K-nitrate gave significantly higher values than K-chloride. The application time had no significant effect on processing grade and total tuber yield or on tuber specific gravity and dry-matter percentage. Values for crisp colour and reducing sugars were within the acceptable range for all treatments. Crisp yields were calculated to be highest and oil percentage of the crisps to be lowest when Ksulphate was applied as K fertilizer. Net income and benefit:cost ratio were highest for K-chloride followed by K-sulphate and lowest for K-nitrate. The study suggests that for crisping potatoes, K-sulphate is more suited than K-chloride, as it not only increased tuber dry-matter percentage and crisp yield, but also decreased crisp oil percentage.

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

A field study was conducted during 2000-2001 and 2001-2002 at the Central Potato Research Institute Campus, Modipuram, India, in order to increasing the processing-grade tuber yield of India's first ever developed processing potato cultivars, Kufri Chipsona-1 and Kufri Chipsona-2. Tuber yield and post-harvest quality characteristics were evaluated in response to five N levels (0, 90, 180, 270 and 360 kg N ha -1 ). The crop growth traits (stem number, plant height and compound leaf number) responded positively to N application, whereas the effect of N fertilization on processing-grade tuber number, total tuber number per ha and tuber number per plant was quadratic. There was a steady increase in tuber weight per plant, processing-grade tuber yield, total tuber yield and biomass yield in response to N application. Kufri Chipsona-1 produced a 23.6% higher tuber yield per plant than Kufri Chipsona-2. Agronomic N use efficiency decreased linearly with increased N doses. Specific gravity and tuber dry matter percentage responded positively to N application, while crisps colour (at harvest and after storage) and reducing sugars remained unaffected. Cultivar was the major factor that influenced the tuber quality parameters (specific gravity, crisps colour). Higher values of these quality traits were observed in Kufri Chipsona-2 as compared to Kufri Chipsona-1. Net income and benefit cost ratio (B:C) indicated that Chipsona cultivars should be fertilized with 270 kg N ha -1 for realizing higher processing-grade yields and desirable quality tubers.

The effects of jasmonic acid (JA) and methyl jasmonate (MeJA) on potato (Solanum tuberosum L.) tuberization were studied in relation to cytokinins using single-node cuttings (SNCs) in vitro. In three potato cultivars differing in maturity levels, JA or MeJA (0.0, 2.5, 5.0, 7.5 and 10.0 lM) action was examined with N 6 -benzyladenine (BA: 0.0, 22.0 and 44.0 lM) under optimum tuber-inducing treatment with 80 g L -1 sucrose. Although jasmonates had a stimulatory role in root growth from SNCs, BA inhibited the root growth and antagonized the jasmonates action on root growth promotion. There was a strong inhibitory effect of BA on stoloniferous shoot growth, and in combination with jasmonates it could exert a much pronounced inhibitory effect. Jasmonates did not have any role in tuber induction in terms of tuber number and sourcesink coordination (harvest index), rather their effects were counteracted in the presence of 22.0 lM BA in early cultivar. However, they had a promoting effect on tuber growth after induction in early cultivar, possibly due to its lower levels of endogenous gibberellins.Cytokinins detrimentally affected the tuber growth, and antagonized the jasmonates action irrespective of the maturity levels of the cultivars. It could, however, increase the tuber dry matter concomitant with a higher accumulation of starch. The promoting effect of jasmonates on tuber dry matter and starch accumulation was apparent only in early cultivar. There were significant interactions between cytokinin and jasmonates for accumulation of reducing and total sugars in tubers. The results show that exogenous cytokinins antagonize the jasmonates action, and the correlated effects of these two growth hormones interact with the maturing time of the cultivar during potato tuber formation in vitro.

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