Emergence,evolution, and vaccine production approaches of SARS-CoV-2 virus: Benefits of getting vaccinated and common questions

The emergence of coronavirus disease 2019 (COVID-19) pandemic in Wuhan city, China at the end of 2019 made it urgent to identify the origin of the causal pathogen and its molecular evolution, to appropriately design an effective vaccine. This study analyzes the evolutionary background of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS-2) in accordance with its close relative SARS-CoV (SARS-1), which was emerged in 2002. A comparative genomic and proteomic study was conducted on SARS-2, SARS-1, and Middle East respiratory syndrome coronavirus (MERS), which was emerged in 2012. In silico analysis inferred the genetic variability among the tested viruses. The SARS-1 genome harbored 11 genes encoding 12 proteins, while SARS-2 genome contained only 10 genes encoding for 10 proteins. MERS genome contained 11 genes encoding 11 proteins. The analysis also revealed a slight variation in the whole genome size of SARS-2 comparing to its siblings resulting from sequential insertions and deletions (indels) throughout the viral genome particularly ORF1AB, spike, ORF10 and ORF8. The effective indels were observed in the gene encoding the spike protein that is responsible for viral attachment to the angiotensin-converting enzyme 2 (ACE2) cell receptor and initiating infection. These indels are responsible for the newly emerging COVID-19 variants αCoV, βCoV, γCoV and δCoV. Nowadays, few effective COVID-19 vaccines developed based on spike (S) glycoprotein were approved and become available worldwide. Currently available vaccines can relatively prevent the spread of COVID-19 and suppress the disease. The traditional (killed or attenuated virus vaccine and antibody-based vaccine) and innovated vaccine production technologies (RNA- and DNA-based vaccines and viral vectors) are summarized in this review. We finally highlight the most common questions related to COVID-19 disease and the benefits of getting vaccinated.     

Impact of catch-up vaccination on aluminum exposure due to new laws and post social distancing

BackgroundThe COVID-19 pandemic has placed significant stressors on the medical community and on the general public. Part of this includes patients skipping well-child visits to reduce risk of exposure to SARS-CoV-2 virus. Published estimates of the duration of whole-body aluminum (Al) toxicity from vaccines in infants from birth to six months indicate that CDC's recommended vaccination schedule leads to unacceptably long periods of time in which infants are in aluminum toxicity (as measured by %AlumTox).MethodsWe utilize these established clearance and accumulation models to calculate expected per-body-weight whole-body toxicity of aluminum from vaccines considering for children of all ages under CDC's Catch-Up schedule from birth to ten years, assuming social distancing for 6 months. Our updated Pediatric Dose Limit (PDL) model assumes a linear improvement in renal function from birth to two years.ResultsOur results indicate that due diligence in considering alternative spacing and use of non-aluminum containing vaccines when possible will reduce whole body toxicity and may reduce risk of morbidity associated with exposure to aluminum.ConclusionsWhile reduction or elimination of aluminum exposure from all sources is always a good idea, our results indicate that careful consideration of expected aluminum exposures during regular and Catch-Up vaccination is found to be especially important for infants and children below 2 years of age. We urge caution in the mass re-starting of vaccination under CDC’s Catch-Up schedule for children under 12 months and offer alternative strategies to minimize per-day/week/month exposure to aluminum hydroxide following the COVID-19 period of isolation.     

Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3-5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.     

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.     

Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine’s performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin’s psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.     

基因疫苗导入技术研究进展

基因疫苗积极的临床结果证明了,基因免疫是一种有效的临床免疫模式。虽然,喷射注射法的精确作用机制还不太清楚,但临床前研究表明,在皮肤内直接打靶抗原呈递细胞可有效地增强免疫反应。另外,局部给药法和树突细胞体外加载抗原的实验结果显示,直接打靶抗原呈递细胞可放大、控制和调节预防及治疗性基因疫苗的免疫结果。尽管基因枪有许多令人鼓舞的优点,但由于价格和便利性的障碍,它是否能商业化还不能确定。利用基因法治疗和预防疾病所涉及的安全性对基因疫苗要求更严格。这要有可控的质粒导入系统和组织特异性表达系统。     

HPV16l1基因的克隆及其在真核细胞中的表达

克隆并表达人乳头瘤病毒16型(HPV16)晚期基因l1,以期为研制防治宫颈癌的DNA疫苗奠定基础。本实验采用PCR方法从质粒p16L1BN1中获得HPV16l1基因片段,利用基因重组技术,将其克隆至含巨细胞病毒(CMV)启动子的真核表达载体中,核酸序列鉴定HPV16l1基因真核表达质粒构建成功,再用脂质体介导基因转染7721人肝癌细胞。转化阳性细胞经SDS-PAGE显示在分子量大约为55kDa的位置出现一条特异性条带,与HPV16L1分子量大小相符。表达产物经Western blotting分析:能与HPV16L1单克隆抗体特异结合。真核表达质粒pcDNA3-HPV16L1构建成功并能在真核细胞7721中有效表达,为下一步进行动物DNA免疫实验奠定了基础。     

Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV‐vaccines and live white spot syndrome virus

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate ‘vaccines’, WSSV envelope protein VP28 and formalin‐inactivated WSSV, can provide short‐lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live‐WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV‐intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune‐related, intracellular organelle part, intracellular calcium‐binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV‐intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.     

Evaluation of efficacy of stabilizers on the thermostability of live attenuated thermo-adapted <Emphasis Type="Italic">Peste des petits ruminants</Emphasis> vaccines

In this study, thermo-adapted (Ta) PPR vaccines were assessed for their stability at 25, 37, 40, 42 and 45°C in lyophilized form using two extrinsic stabilizers {lactalbumin hydrolysate-sucrose (LS) and stabilizer E} and in reconstituted form with the diluents (1 mol/L MgSO₄ or 0.85% NaCl). The lyophilized vaccines showed an expiry period of 24–26 days at 25°C, 7–8 days at 37°C and 3–4 days at 40°C. LS stabilizer was superior at 42°C with a shelf-life of 44 h, whereas in stabilizer E, a 40 h shelf-life with a comparable half-life was observed. At 45°C, the half-life in stabilizer E was better than LS and lasted for 1 day. Furthermore, the reconstituted vaccine maintained the titre for 48 h both at 4°C and 25°C and for 24–30 h at 37°C. As both the stabilizers performed equally well with regard to shelf-life and half-life, the present study suggests LS as stabilizer as a choice for lyophilization with 0.85% NaCl diluent, because it has better performance at higher temperature. These Ta vaccines can be used as alternatives to existing vaccines for the control of the disease in tropical countries as they are effective in avoiding vaccination failure due to the breakdown in cold-chain maintenance, as this vaccine is considerably more stable at ambient temperatures.