Recombinant Adenovirus Vaccine Research Articles

Intranasal Immunization with a Recombinant Adenovirus Encoding Multi-Stage Antigens of Mycobacterium tuberculosis Preferentially Elicited CD8+ T Cell Immunity and Conferred a Superior Protection in the Lungs of Mice than Bacillus Calmette-Guerin.

The development of a tuberculosis (TB) vaccine is imperative. Employing multi-stage Mycobacterium tuberculosis (Mtb) antigens as targeted antigens represents a critical strategy in establishing an effective novel TB vaccine. In this investigation, we evaluated the immunogenicity and protective efficacy of a recombinant adenovirus vaccine expressing two fusion proteins, Ag85B-ESAT6 (AE) and Rv2031c-Rv2626c (R2), derived from multi-stage antigens of Mtb via intranasal administration in mice. Intranasal delivery of Ad-AE-R2 induced both long-lasting mucosal and systemic immunities, with a preferential elicitation of CD8+ T cell immunity demonstrated by the accumulation and retention of CD8+ T cells in BALF, lung, and spleen, as well as the generation of CD8+ TRM cells in BALF and lung tissues. Compared to subcutaneous immunization with Bacillus Calmette-Guerin (BCG), Ad-AE-R2 provided superior protection against high-dose intratracheal BCG challenge, specifically within the lungs of mice. Our findings support the notion that empowering T cells within the respiratory mucosa is crucial for TB vaccine development while highlighting targeting CD8+ T cell immunity as an effective strategy for optimizing TB vaccines and emphasizing that eliciting systemic memory immunity is also vital for the successful development of a TB mucosal vaccine. Furthermore, our results demonstrate that the BCG challenge serves as a convenient and efficient method to evaluate candidate vaccine efficacy.

Intranasal delivery of a recombinant adenovirus vaccine encoding the PEDV COE elicits potent mucosal and systemic antibody responses in mice.

Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus that causes substantial economic loss to the global pig industry. The emergence of PEDV variants has increased the need for new vaccines, as commercial vaccines confer inferior protection against currently circulating strains. It is well established that the induction of mucosal immunity is crucial for PEDV vaccines to provide better protection against PEDV infection. In this study, we constructed a recombinant adenovirus expressing the core neutralization epitope (COE) of G2b PEDV based on human adenovirus serotype 5 (Ad5). We evaluated the effects of different administration routes and doses of vaccine immunogenicity in Balb/c mice. Both intramuscular (IM) and intranasal (IN) administration elicited significant humoral responses, including COE-specific IgG in serum and mucosal secretions, along with serum-neutralizing antibodies. Moreover, IN delivery was more potent than IM in stimulating IgA in serum and mucosal samples and in dampening the immune response to the Ad5 vector. The immune response was stronger after high versus low dose IM injection, whereas no significant difference was observed between high and low IN doses. In summary, our findings provide important insights for developing novel PEDV vaccines.IMPORTANCEPorcine epidemic diarrhea (PED) is a highly contagious disease that has severe economic implications for the pork industry. Developing an effective vaccine against PEDV remains a necessity. Here, we generated a recombinant adenovirus vaccine based on Ad5 to express the COE protein of PEDV (rAd5-PEDV-COE) and systematically evaluated the immunogenicity of the adenovirus-vectored vaccine using different administration routes (intramuscular and intranasal) and doses in a mouse model. Our results show that rAd5-PEDV-COE induced potent systemic humoral response regardless of the dose or immunization route. Notably, intranasal delivery was superior to induce peripheral and mucosal IgA antibodies compared with intramuscular injection. Our data provide valuable insights into designing novel PEDV vaccines.

Immunogenicity Analysis and Identification of Potential T-Cell Epitopes in C129R Protein of African Swine Fever Virus.

The highly conserved C129R protein of AFSV was utilized in the development of an ASFV recombinant adenovirus vaccine, demonstrating strong immunogenicity. In this study, we immunized 6-week-old female C57BL/6J mice via subcutaneous injection with 10 μg of purified C129R protein. Humoral and cellular immune effects were assessed using ELISA, flow cytometry, and ELISpot assays. Additionally, 19 peptides of the C129R protein were synthesized and screened for the use of bioinformatics. Positive T-cell epitopes were screened using ELISpot. The results indicated a higher proportion of CD4+ and CD8+ T lymphocytes in immunized mice compared to control mice. ELISA analysis revealed a serum titer of approximately 1:1, 638, 400 in the experimental group of mice. Additionally, peptides C11(53-61aa), C14(81-89aa), C16(97-105aa), and C18(116-124aa) from the C129R protein were able to activate mice spleen lymphocytes to produce IFN-γ. These findings suggest that the C129R protein significantly enhances both humoral and cellular immunity in immunized mice. Moreover, peptides C11, C14, C16, and C18 may serve as potential T-cell epitopes for the C129R protein. These results lay the groundwork for the further exploration of ASFV C129R protein and the identification of novel ASF vaccine antigens.

Application of Multivariate Data Analysis on Historical Recombinant Adenovirus Zoster Vaccine Production Data for Upstream Process Improvements

In recent years, multivariate data analysis (MVDA) has been widely used for process characterization and fault diagnosis in the biopharmaceutical industry. This study aims to investigate the feasibility of using MVDA for the development and scale-up of a perfusion process for HEK293 cell-based recombinant adenovirus zoster vaccine (Ad-HER) production. The Principal Component Analysis (PCA) results suggested comparable performance among the ATF, PATFP, and BFP perfusion systems in benchtop-scale stirred-tank bioreactor (STR). Then a Batch Evolution Model (BEM) was built using representative data from 10 L STR with a BFP system to assess the Ad-HER perfusion process performance at pilot-scale bioreactor (50 L STR and 50 L wave bioreactor). Furthermore, another BEM model and Batch Level Model (BLM) were built to monitor process parameters over time and predict the final adenovirus titer in 50 L wave bioreactor. The loading plot revealed that lactate dehydrogenase activity, viable cell diameter, and base-added during the virus production phase could be used as preliminary indicators of adenovirus yield. Finally, an adenovirus titer of 2.0±0.3×1010 IFU/mL was achieved in the 50 L wave bioreactor with BFP system, highlighting the robustness of the Ad-HER perfusion process at pilot-scale. Overall, this study emphasizes the effectiveness of MVDA as a tool for advancing the understanding of recombinant adenovirus vaccine perfusion production process development and scale-up.

Simultaneous Expression of Chicken Granulocyte Monocyte Colony-Stimulating Factor and the Hemagglutinin-Neuraminidase Epitope of the Virulent Newcastle Disease Virus Genotype VII C22 Strain in a Functional Synthetic Recombinant Adenovirus as a Genotype-Matched Vaccine with Potential Antiviral Activity.

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-β, IFN-γ, interleukin-1β (IL-1β), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.

Impact of vaccination against COVID-19 on the outcomes of in vitro fertilization-embryo transfer: a retrospective cohort study.

Vaccination against coronavirus disease 2019 (COVID-19) has become the primary approach in the fight against the spread of COVID-19. Studies have shown that vaccination against COVID-19 has adverse effects, particularly on human reproductive health, despite the fact that vaccination rates are still on the rise. However, few studies have reported whether vaccination affects the outcome of in vitro fertilization-embryo transfer (IVF-ET) or not. In this study, we compared the outcome of IVF-ET and the development of follicles and embryos between vaccinated and unvaccinated groups. A single-center retrospective cohort study of 10,541 in vitro fertilization (IVF) cycles was conducted from June 2020 to August 2021. 835 IVF cycles with a history of vaccination against COVID-19 and 1670 IVF cycles that served as negative controls were selected and analyzed utilizing the Matchlt package of R software (http://www.R-project.org/) and the nearest neighbor matching algorithm for propensity-matched analysis at a 1:2 ratio. The number of oocytes collected in the vaccinated group and the unvaccinated group were 8.00 (0, 40.00) and 9.00 (0, 77.00) (P = 0.073) and the good-quality embryo rates of the two groups were 0.56±0.32 and 0.56±0.31 averagely (P = 0.964). Clinical pregnancy rates for the vaccinated group and unvaccinated group were 42.4% (155/366) and 40.2% (328/816) (P = 0.486) and biochemical pregnancy rates were 7.1% (26/366) and 8.7% (71/816) (P = 0.355). Two other factors were analyzed in this study; vaccination among different genders and different types (inactivated vaccine or recombinant adenovirus vaccine) showed no statistically significant effect on the above outcomes. In our findings, vaccination against COVID-19 showed no statistically significant effect on the outcomes of IVF-ET and the development of follicles and embryos, nor did the gender of the vaccinated person or the formulation of vaccines showed significant effects.

Intranasal COVID-19 vaccine: a modern vaccination approach.

Dear Editor, The emergence of severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) which was originally reported to WHO in December 2019 resulted in the coronavirus illness and it was subsequently declared a global coronavirus disease 2019 (COVID-19) pandemic in March 2020. Recurring disease outbreaks repeatedly overloaded the public health sector, infecting over 613 million people and killing more than 6.5 million as of September 20221. As new strains emerge and natural immunity wanes, the population, particularly immunocompromised individuals, stand an increased risk of severe sickness and death due to this disease. Various research advances paved the way for developing multiple COVID-19 vaccines which have been approved to treat and prevent COVID-19. Currently, available COVID-19 vaccines including mRNA vaccines, recombinant adenovirus vaccines, and recombinant protein vaccines that have received Emergency Use Authorization (EUA) and have been subsequently used to immunize millions of people are all administered through intramuscular injection2. Intramuscular vaccines have been designed to induce antibody-mediated and cell-mediated immune responses and minimal to no detectable mucosal immunity at the site of infection. Since the nose and upper respiratory tract are the primary routes for inhalation pathogens like SARS-CoV-2 and are more susceptible to infection,3 the vaccine response generated after intramuscular immunization can leave the upper respiratory tract vulnerable to viral replication and dissemination because these vaccines are inefficient in stimulating immunoglobulin A secretion in mucosal cells2. Alternatively, it has been found recently that the intranasal (IN) vaccines for COVID-19 have shown a promising ability to induce sterilizing mucosal immunity against mucosal pathogens apart from inducing an antibody-mediated immune response and a robust cell-mediated immunity2. A single dose of effective SARS-CoV-2 vaccine via IN route boosts mucosal immunoglobulin A and T-cell responses in both upper and lower respiratory tract, providing almost complete prevention against viral infection, multiplication, and reducing virus shedding via nasal mucosa that acts as a first-line barrier to the virus entrance before spreading into the lungs2. This immediate protection at the site of infection by restricting the viral replication and clearance of SARS-CoV-2 infection reflects the potency of IN COVID-19 vaccines in the alleviation of seeding new cases of COVID-19 amidst the pandemic; hence, preventing even mild cases of illness and inhibiting the virus transmission to other people. In addition, these vaccines are minimally invasive, have a needle-free mode of administration, and are usually in the form of a nasal spray2. Owing to the easy transportation and storage, these vaccines are also more convenient to use2. However, an essential consideration for IN COVID-19 vaccine is its safety. An extremely rare risk of reversion of live attenuated virus into replicating state, regaining its ability to cause disease in the individual has been observed4. With that, the effectiveness of the vaccine is determined by the immunization type chosen and the addition of essential adjuvants. Phase I, II, and III clinical trials were performed to assess the efficacy of IN COVID-19 immunization by two companies; CanSino Biologics, a Chinese pharmaceutical company, and Bharat Biotech International, a biotechnology company headquartered in India, which received approval in their respective countries for its use after gaining successful results5. These companies have created ‘viral vector’ vaccines that use a harmless adenovirus to deliver the genetic material of the SARS-CoV-2 into host cells. Given the severity of the situation where the healthcare systems are challenged globally due to increased COVID-19-related hospitalizations, it is critical to continue developing effective and reliable COVID-19 vaccines in present times. In the context of respiratory pathogens like SARS-CoV-2, IN vaccines’ pros conceivably outweigh their cons, along with offering both localized protection at the site of infection and activating systemic responses as compared with intramuscular vaccines eliciting only systemic responses. Large-scale clinical studies and trials should be conducted to gather further evidence to prove its effectiveness so that the availability of IN COVID-19 vaccines may be made feasible. This vaccine might also serve as an alternative to limited and inadequate vaccine supplies as well as a booster to intramuscular vaccines. Immunization with IN COVID-19 vaccines will hopefully help deal with the persistent COVID-19 pandemic and potential viral contagious diseases that might arise in the future. Ethics approval Not applicable. Sources of funding None. Authors’ contribution All authors contributed equally. Conflicts of interest disclosure The authors declare that they have no financial conflict of interest with regard to the content of this report. Research registration Unique Identifying number (UIN) None. Guarantor Govinda Khatri.

Development of a recombinant adenovirus-vectored vaccine against both infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss)

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.

Fight against the delta variant! Where does Pakistan stand?

Dear Madam, Since its first appearance in India in December 2020, the B.1.617.2 (delta) variant has spread across 85 countries as of yet. Its rapid proliferation has enforced WHO to classify the strain as a “variant of concern”. Equipped with 10 spike protein mutations (T19R, (G142D*), 156del, 157del, R158G, L452R, T478K, D614G, P681R, D950N), especially the P681R mutation at the cleavage site S1-S2,1 the virus now has the ability to replicate and transmit at a rate much faster than the original SARS-CoV-2. This is evident from the fact that it is detectable in infected individuals just under 4 days after it’s contraction, with a viral load 1,260 times higher, as compared to 6 days with the earliest form.2 Researchers are uncertain regarding the efficacy of existing vaccines against the delta strain. A recent preliminary study analysed the efficacy of BNT162b2 (Pfizer) and ChAdOx1 nCoV-19 (AstraZeneca) against the mutated version. The study revealed markedly lower effectiveness after one dose, and moderate effectiveness after two doses among people infected with delta variant for both the vaccines.3 The recent finding is daunting for Pakistan as the nation has seen a rapid surge in delta variant cases. In Karachi alone, the positivity ratio has inflated up to an alarming level of 23.6% with the major hospitals operating at full capacity.4 Although the process of vaccination is ongoing, still 20202951(20 million) individuals remain partially vaccinated,5 while the rest of the population remains unvaccinated which poses a serious concern for Pakistan since such individuals, as stated earlier, are at a greater risk of contracting the delta variant.3 Considering that the virus will continue to mutate, it is imperative that preventive guidelines including the use of a mask, social distancing must be practiced and travel restrictions must be imposed in order to curtail the spread. Additionally, booster shots can be administered to increase the efficacy of these vaccines. Moreover, a study conducted on mice evaluated the response of recombinant chimpanzee adenovirus (AdC7) vaccine expressing S, receptor-binding domain (RBD) or tandem-repeat dimeric RBD (RBD-tr2) (AdC7-RBD-tr2) administered via intramuscular (systemic) and intranasal (mucosal) route. This vaccine exhibited a greater neutralising effect on the delta variant than the existing vaccines. The intranasal administration produced mucosal immunity with neutralizing activity in Broncho alveolar lavage fluid thus offering greater protection to the respiratory tract, a quintessential goal to terminate SARS-CoV-2 transmission.6

Safety of SARS-CoV-2 vaccines in patients with chronic myeloid leukemia: a multicenter survey in China.

Safety of SARS-CoV-2 vaccines in patients with chronic myeloid leukemia: a multicenter survey in China.

Adenovirus vaccine therapy with CD137L promotes CD8+ DCs-mediated multifunctional CD8+ T cell immunity and elicits potent anti-tumor activity

Renal carcinoma progresses aggressively in patients with metastatic disease while curative strategies are limited. Here, we constructed a recombinant non-replicating adenovirus (Ad) vaccine encoding an immune activator, CD137L, and a tumor antigen, CAIX, for treating renal carcinoma. In a subcutaneous tumor model, tumor growth was significantly suppressed in the Ad-CD137L/CAIX vaccine group compared with the single vaccine group. The induction and maturity of CD11C+ and CD8+CD11C+ dendritic cell (DC) subsets were promoted in Ad-CD137L/CAIX co-immunized mice. Furthermore, the Ad-CD137L/CAIX vaccine elicited stronger tumor-specific multifunctional CD8+ T cell immune responses as demonstrated by increased proliferation and cytolytic function of CD8+ T cells. Notably, depletion of CD8+ T cells greatly compromised the effective protection provided by Ad-CD137L/CAIX vaccine, suggesting an irreplaceable role of CD8+ T cells for the immunopotency of the vaccine. In both lung metastatic and orthotopic models, Ad-CD137L/CAIX vaccine treatment significantly decreased tumor metastasis and progression and increased the induction of tumor-specific multifunctional CD8+ T cells, in contrast to treatment with the Ad-CAIX vaccine alone. The Ad-CD137L/CAIX vaccine also augmented the tumor-specific multifunctional CD8+ T cell immune response in both orthotopic and metastatic models. These results indicated that Ad-CD137L/CAIX vaccine elicited a potent anti-tumor activity by inducing CD8+DC-mediated multifunctional CD8+ T cell immune responses. The potential strategy of CD137L-based vaccine might be served as a novel treatment for renal carcinoma or other malignant tumors.

COVID-19 Vaccine Hesitancy and Early Adverse Events Reported in a Cohort of 7,881 Italian Physicians.

The COVID-19 vaccination campaign began in Italy at the end of December 2020, with the primary aim of immunizing healthcare professionals, using the EMA approved mRNA vaccines (Comirnaty® by Pfizer/BioNTech; mRNA-1273 by Moderna) and recombinant adenoviral vaccine (Vaxzevria® by AstraZeneca). The study aimed at evaluating the prevalence and motivations underlying Vaccine Hesitancy, as well as the incidence and type of adverse events associated with COVID-19 vaccination. Cross-sectional study. Data were collected January 1st to 28th 2021 using a purposely created online self-administered questionnaire from a selected cohort of Italian physicians. Overall, 7,881 questionnaires were analyzed: 6,612 physicians had received one dose, and 1,670 two doses of Comirnaty®; 30 had received one dose of mRNA-1273. Vaccine Hesitancy rate was 3.6%; it correlated with prior SARS-CoV-2 infection, diabetes, Adverse Eventss at previous vaccinations and refusal of 2020 flu vaccine, and was mainly motivated by concerns about vaccine Adverse Events. Typical Adverse Events were pain/itching/paresthesia at the inoculation site, followed by headache, fever, fatigue and myalgia/arthralgia occurring more frequently after the second dose (77.8 vs 66.9%; p<0.001), and in subjects with a prior SARS-CoV-2 infection. Adherence to COVID-19 vaccination is high among physicians. Adverse Events are typically mild and more frequent in people with a prior SARS-CoV-2 infection.

Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages

SummaryA quarter of all seasonal influenza cases are caused by type B influenza virus (IBV) that also dominates periodically. Here, we investigated a recombinant adenovirus vaccine carrying a synthetic HA2 representing the consensus sequence of all IBV hemagglutinins. The vaccine fully protected mice from lethal challenges by IBV of both genetic lineages, demonstrating its breadth of protection. The protection was not mediated by neutralizing antibodies but robust antibody-dependent cellular cytotoxicity and cell-mediated immune responses. Complete protection of the animals required the entire codon-optimized HA2 sequence that elicited a balanced immune response, whereas truncated vaccines without either the fusion peptide or the transmembrane domain reduced the efficacy of protection. Finally, the vaccines did not demonstrate any sign of disease exacerbation following lung pathology and morbidity monitoring. Collectively, these data suggest that it could be worth further exploring this prototype universal vaccine because of its considerable efficacy, safety, and breadth of protection.

The Next Set of COVID-19 Vaccines: Leveraging New Development Platforms to Increase Access for More People Around the World

The approval of the coronavirus disease 2019 (COVID-19) mRNA vaccines brought much optimism to efforts to end the pandemic. A recombinant adenovirus vaccine recently received emergency use authorization, and several other vaccines are likely to follow. These vaccines all use relatively new vaccine production platforms to produce the severe acute respiratory syndrome coronavirus 2 Spike protein. This review discusses how these platforms work, what advantages they offer, and the gaps that remain in public health efforts to control the COVID-19 pandemic. (Clin Ther. 2021;43:702–710) © 2021 Elsevier HS Journals, Inc.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals low levels of viral backbone gene transcription alongside very high levels of SARS-CoV-2 S glycoprotein gene transcription

BackgroundChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene.MethodsWe used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine.ResultsThe expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells, but in A549 cells, there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells.ConclusionsOverall, the ChAdOx1 nCoV-19 vaccine’s transcriptomic and proteomic repertoire in cell culture is as expected. The combined transcriptomic and proteomics approaches provide a detailed insight into the behaviour of this important class of vaccine using state-of-the-art techniques and illustrate the potential of this technique to inform future viral vaccine vector design.

Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat spike protein RBD protects mice against COVID-19

ABSTRACT A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus-vectored vaccines expressing spike (S) protein have been approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD), or tandem-repeat dimeric RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular immune responses. AdC7-RBD-tr2 showed higher antibody responses compared to either AdC7-S or AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. AdC7-RBD-tr2-elicted sera preserved the neutralizing activity against the circulating variants, especially the Delta variant. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.

Freeze-Drying Formulations Increased the Adenovirus and Poxvirus Vaccine Storage Times and Antigen Stabilities.

Successful vaccines induce specific immune responses and protect against various viral and bacterial infections. Noninactivated vaccines, especially viral vector vaccines such as adenovirus and poxvirus vaccines, dominate the vaccine market because their viral particles are able to replicate and proliferate in vivo and produce lasting immunity in a manner similar to natural infection. One challenge of human and livestock vaccination is vaccine stability related to the antigenicity and infectivity. Freeze-drying is the typical method to maintain virus vaccine stability, while cold chain transportation is required for temperatures about 2 °C–8 °C. The financial and technological resource requirements hinder vaccine distribution in underdeveloped areas. In this study, we developed a freeze-drying formula consisting of bovine serum albumin (BSA), l-glutamic acid (L-Glu), polyethylene glycol (PEG), and dextran (DEX) to improve the thermal stability and activity of viral vaccines, including vaccinia recombinant vaccine (rTTV-OVA) and adenovirus vaccine (Ad5-ENV). We compared a panel of five different formulations (PEG: DEX: BSA: L-GLU = 50:9:0:0(#1), 50:5:4:0(#2), 50:10:9:0(#3), 50:0:0:9(#4), and 50:1:0:8(#5), respectively) and optimized the freeze-drying formula for rTTV-OVA and Ad5-ENV. We found that the freeze-drying formulations #2 and #3 could maintain rTTV-OVA infectivity at temperatures of 4 °C and 25 °C and that rTTV-OVA immunogenicity was retained during lyophilization. However, formulations #4 and #5 maintained Ad5-ENV infectivity under the same conditions, and Ad5-ENV immunogenicity had maximum retention with freeze-drying formulation #4. In summary, we developed new freeze-drying formulations that increased virus vaccine storage times and retained immunogenicity at an ambient temperature.

Heterologous prime-boost: an important candidate immunization strategy against Tembusu virus

BackgroundTembusu virus (TMUV), a newly emerging pathogenic flavivirus, spreads rapidly between ducks, causing massive economic losses in the Chinese duck industry. Vaccination is the most effective method to prevent TMUV. Therefore, it is urgent to look for an effective vaccine strategy against TMUV. Heterologous prime-boost regimens priming with vaccines and boosting with recombinant adenovirus vaccines have been proven to be successful strategies for protecting against viruses in experimental animal models.MethodsIn this study, heterologous and homologous prime-boost strategies using an attenuated salmonella vaccine and a recombinant adenovirus vaccine expressing prM-E or the E gene of TMUV were evaluated to protect ducks against TMUV infection for the first time, including priming and boosting with the attenuated salmonella vaccine, priming and boosting with the recombinant adenovirus vaccine, and priming with the attenuated salmonella vaccine and boosting with the recombinant adenovirus vaccine. Humoral and cellular immune responses were detected and evaluated. We then challenged the ducks with TMUV at 12 days after boosting to assay for clinical symptoms, mortality, viral loads and histopathological lesions after these different strategies.ResultsCompared with the homologous prime-boost strategies, the heterologous prime-boost regimen produced higher levels of neutralizing antibodies and IgG antibodies against TMUV. Additionally, it could induce higher levels of IFN-γ than homologous prime-boost strategies in the later stage. Interestingly, the heterologous prime-boost strategy induced higher levels of IL-4 in the early stage, but the IL-4 levels gradually decreased and were even lower than those induced by the homologous prime-boost strategy in the later stage. Moreover, the heterologous prime-boost strategy could efficiently protect ducks, with low viral titres, no clinical symptoms and histopathological lesions in this experiment after challenge with TMUV, while slight clinical symptoms and histopathological lesions were observed with the homologous prime-boost strategies.ConclusionsOur results indicated that the heterologous prime-boost strategy induced higher levels of humoral and cellular immune responses and better protection against TMUV infection in ducks than the homologous prime-boost strategies, suggesting that the heterologous prime-boost strategy is an important candidate for the design of a novel vaccine strategy against TMUV.

Immunogenicity of an Adenoviral Vector Vaccine (rAd‐MOMP‐CpG) Against Chlamydia Trachomatis

Chlamydia trachomatis (C. trachomatis) infection is categorized as an important causes of sexually transmitted diseases in humans. Currently, there is no approved vaccine against C. trachomatis. This study was conducted to develop and evaluate the immunogenicity of a recombinant adenovirus (rAd‐MOMP‐CpG) vaccine against C. trachomatis in mice. The vaccine was constructed using adenovirus type 5 based on the major outer membrane protein (MOMP) antigen of C. trachomatis. Sixty mice were divided into 4 groups. In group 1, mice received rAd‐MOMP‐CpG (5×108 PFU) intranasal, 3 times at 2‐weeks intervals. Mice in groups 2, 3 and 4 received equivalent volume of adenovirus vector, CpG 1826, and PBS, respectively. Splenic cytokine levels and mRNA expression levels of IFNg and IL‐4 were determined by ELISA and qPCR, respectively. Furthermore, serum specific murine anti‐MOMP IgG levels were determined by quantitative ELISA. The constructed rAd‐MOMP‐CpG vaccine provoked a Chlamydia‐specific T helper type 1 response. There was a significant (P = 0.047) splenic IFNg stimulation in vaccinated group in comparison to the vector‐control group. The splenic IL‐4 expression remained at minimal stimulation levels across time points. In addition, the vaccine induced significant increase (P = 0.006) of delayed humoral anti‐MOMP IgG response compared to control groups. A recombinant adenovirus vaccine against C. trachomatis could provide important clues toward a vaccine development for C. trachomatis serovar protection in humans. However, further in vitro and in vivo studies are warranted to determine the most effective dosage, vaccine efficacy and safety.Support or Funding InformationJordan University of Science and Technology, Deanship of Research (grant number: 20140275).

Recombinant adenovirus carrying a core neutralizing epitope of porcine epidemic diarrhea virus and heat-labile enterotoxin B of Escherichia coli as a mucosal vaccine.

Porcine epidemic diarrhea virus (PEDV) targets the intestinal mucosa in pigs. To protect against PEDV invasion, a mucosal vaccine is utilized effectively. In this study, we generated a recombinant adenovirus vaccine encoding the heat-labile enterotoxin B (LTB) and the core neutralizing epitope (COE) of PEDV (rAd-LTB-COE). The fusion protein LTB-COE was successfully expressed by the recombinant adenovirus in HEK293 cells, and the immunogenicity of the vaccine candidate was assessed in BALB/c mice and piglets. Three intramuscular or oral vaccinations with rAd-LTB-COE at two-week intervals induced robust humoral and mucosal immune responses. Moreover, a cell-mediated immune response was promoted in immunized mice, and the neutralizing antibody inhibited both the vaccine strain and the emerging PEDV isolate. Immunization experiments in piglets revealed that rAd-LTB-COE was immunogenic and induced good immune responses in piglets. Further studies are required to evaluate the efficacy of rAd-LTB-COE against a highly virulent PEDV challenge.