Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine:"Adenovirus Vaccine Within an Adenovirus Vector"

Human adenoviruses(HAd Vs)are highly contagious and result in large number of acute respiratory disease(ARD)cases with severe morbidity and mortality.Human adenovirus type 3(HAd V-3)is the most common type that causes ARD outbreaks in Asia,Europe,and the Americas.However,there is currently no vaccine approved for its general use.The hexon protein contains the main neutralizing epitopes,provoking strong and lasting immunogenicity.In this study,a novel recombinant and attenuated adenovirus vaccine candidate against HAd V-3 was constructed based on a commercially-available replication-defective HAd V-5 gene therapy and vaccine vector.The entire HAd V-3 hexon gene was integrated into the E1 region of the vector by homologous recombination using a bacterial system.The resultant recombinants expressing the HAd V-3 hexon protein were rescued in AD293 cells,identified and characterized by RT-PCR,Western blots,indirect immunofluorescence,and electron microscopy.This potential vaccine candidate had a similar replicative efficacy as the wild-type HAd V-3 strain.However,and importantly,the vaccine strain had been rendered replication-defective and was incapable of replication in A549 cells after more than twentygeneration passages in AD293 cells.This represents a significant safety feature.The mice immunized both intranasally and intramuscularly by this vaccine candidate raised significant neutralizing antibodies against HAd V-3.Therefore,this recombinant,attenuated,and safe adenovirus vaccine is a promising HAd V-3 vaccine candidate.The strategy of using a clinically approved and replication-defective HAd V-5 vector provides a novel approach to develop universal adenovirus vaccine candidates against all the other types of adenoviruses causing ARDs and perhaps other adenovirus-associated diseases.

Construction of a bivalent vaccine candidate against HAdV4/HAdV7 based on capsid-display strategy via Red-homologous recombination and counter-selection methodology

Human adenoviruses(HAdvs)are major respiratory pathogens.Specifically,human adenovirus type 4(HAdV4)and human adenovirus type 7(HAdV7)are known for causing fever and pneumonia,with docu-mented cases of fatalities among the population.In recent years,HAdV4/HAdv7 has been implicated in caus-ing substantial outbreaks,leading to increased morbidity in multiple countries.Most HAdV4 and HAdV7 infections have been reported in North America,Asia,Europe,Africa,South America,Oceania,and the Middle East.Most fatalities occurred in North America(the United States)and Asia(China and Singapore).Engineered recombinant adenoviruses have played a crucial role as vaccine vectors.In this study,we con-structed a recombinant adenovirus,Ad4ITRmut-Ad7E3,and evaluated it in vitro and in vivo.We observed that the replication rate of Ad4ITRmut-Ad7E3 was lower than that of the RI-67 strain,indicating that the mutation of inverted terminal repeats(ITRs)weakened the replication ability of HAdV4.Immunization of BALB/c mice with the bivalent Ad4ITRmut-Ad7E3 vaccine strain,administered by intraperitoneal injection and oral gavage,resulted in the elicitation of neutralizing antibodies targeting HAdV4 and HAdv7.This finding not only pro-vides a novel method and technique for the efficient construction of a polyvalent recombinant adenovirus vac-cine candidate against HAdV4 and HAdv7 but also against other prevalent adenovirus serotypes such as HAdV3,HAdV11,HAdV14,and HAdv55,from various regions.

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 and25 °C and that r TTV-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.

Specific cellular immune responses in mice immunized with DNA,adeno-associated virus and adenoviral vaccines of Epstein-Barr virus-LMP2 alone or in combination

Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.