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There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in anin vitroACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel®were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel®containing formulation and possibly in combination with other immunostimulants.

Chronic Chagasic cardiomyopathy (CCC) is a severe clinical manifestation that develops in 30%–40% of individuals chronically infected with the protozoal parasite Trypanosoma cruzi and is thus an important public health problem. Parasite persistence during chronic infection drives pathologic changes in the heart, including myocardial inflammation and progressive fibrosis, that contribute to clinical disease. Clinical manifestations of CCC span a range of symptoms, including cardiac arrhythmias, thromboembolic disease, dilated cardiomyopathy, and heart failure. This study aimed to investigate the role of signal transducer and activator of transcription-3 (STAT3) in cardiac pathology in a mouse model of CCC. STAT3 is a known cellular mediator of collagen deposition and fibrosis. Mice were infected with T. cruzi and then treated daily from 70 to 91 days post infection (DPI) with TTI-101, a small molecule inhibitor of STAT3; benznidazole; a combination of benznidazole and TTI-101; or vehicle alone. Cardiac function was evaluated at the beginning and end of treatment by echocardiography. By the end of treatment, STAT3 inhibition with TTI-101 eliminated cardiac fibrosis and fibrosis biomarkers but increased cardiac inflammation; serum levels of interleukin-6 (IL-6), and IFN−γ; cardiac gene expression of STAT1 and nuclear factor-κB (NF-κB); and upregulation of IL-6 and Type I and Type II IFN responses. Concurrently, decreased heart function was measured by echocardiography and myocardial strain. These results indicate that STAT3 plays a critical role in the cardiac inflammatory–fibrotic axis during CCC.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD219-N1C1 subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This vaccine formulation is similar to one that entered advanced phase 3 clinical development in India. We compared the immune response of mice vaccinated with RBD219-N1C1/alum to mice vaccinated with RBD219-N1C1/alum+CpG. We also evaluated mice immunized with RBD219-N1C1/alum+CpG and boosted with RBD219-N1C1/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the RBD219-N1C1/alum formulation, the RBD219-N1C1/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.1 (Kappa) variants. Notably, the sera from mice that received two 7 µg doses of RBD219-N1C1/alum+CpG showed more than 18 times higher neutralizing antibody titers against B.1.351, than the WHO International Standard for anti-SARS-CoV-2 immunoglobulin NIBSC 20/136. Interestingly, a booster dose did not require the addition of CpG to induce this effect. The data reported here reinforces that the RBD219-N1C1/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including three variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.1 (Kappa).

Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi–infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity., Author summary Chagas disease is a chronic infection with Trypanosoma cruzi (T. cruzi) that affects approximately 8 million people worldwide and may cause chronic heart inflammation. The vaccine candidate Tc24-C4 is a recombinant version of the Tc24 protein, which is a flagellar calcium-binding protein expressed by T. cruzi. While animal challenge studies have shown that targeting Tc24 is very promising, it is not fully understood how Tc24 is presented to the immune system. Here, we were able to localize Tc24 in flagellated T. cruzi parasites using a novel Tc24-specific monoclonal antibody. The results showed that Tc24 is not exposed on the outside of the parasite, which suggests that antibodies against Tc24 could not bind parasites during the infection. Then, by analyzing Tc24 expression in T. cruzi—infected host cells over time, we observed that Tc24 expression is reduced after the parasite enters the cells but is restored when parasites escape the host cell again. Our study provides more insights on the location and presence of Tc24 in T. cruzi during infection in the host, and we discuss our current understanding on the mechanisms of how the Tc24 vaccine may work.

BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice.MethodThe RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice.Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 ± 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.