Egg-derived Vaccine Research Articles

Projected impact of a plant-derived vaccine on the burden of seasonal influenza in Canada

ABSTRACT Objective The analysis estimates projected population outcomes resulting from the introduction of a plant-derived influenza vaccine formulated as quadrivalent virus-like particles (QVLP) in Canada. Methods Using Monte Carlo simulations, the number of influenza cases, general practitioner visits, inpatient admissions, intensive care unit (ICU) admissions, and deaths due to influenza-associated illness were estimated under no vaccination, plant-derived QVLP vaccines only, or egg-derived vaccines only. The base case analysis examined the adult Canadian population in two subgroups: 18–64 years of age during the 2017/18 season and 65+ years of age during the 2018/19 season. Efficacy data were obtained from QVLP clinical trials. Vaccine effectiveness data for egg-derived vaccines were calculated from observational studies from the corresponding influenza seasons. Scenario analyses examined the impact of varying absolute vaccine effectiveness or vaccination coverage from base case inputs. Results In the base case analysis, plant-derived QVLP vaccines led to an additional reduction in the burden of influenza over egg-derived vaccines for both population subgroups. In the 18–64 subgroup, QVLP vaccines were associated with 2.63% (48,029; 95% credible interval [Crl]: 42,723–53,336) fewer influenza cases than egg-derived vaccines. In the 65+ subgroup, QVLP vaccines led to 4.82% (27,918; 95% Crl: 25,440–30,397) fewer influenza cases, and reductions in the number of inpatient admissions by 4.77% (1167; 95% CrI: 851–1483) and deaths by 4.75% (326; 95% CrI: 107–546) compared to egg-derived vaccines. Further reductions were observed in scenario analyses considering the potential increase in vaccine coverage. Conclusion Use of plant-derived QVLP influenza vaccines may contribute to greater reductions in influenza cases and influenza-related outcomes, including inpatient admissions and deaths, compared to egg-derived vaccines currently available in Canada.

Process intensification for the production of yellow fever virus-like particles as potential recombinant vaccine antigen.

Yellow fever (YF) is a life-threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000-60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg-derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus-like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence-activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken-tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3-fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one-step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D-structure of the purified VLPs.

Efficacy, immunogenicity, and safety of a plant-derived, quadrivalent, virus-like particle influenza vaccine in adults (18–64 years) and older adults (≥65 years): two multicentre, randomised phase 3 trials

Seasonal influenza remains a substantial public health threat despite the availability of egg-derived and other vaccines. Plant-based manufacturing might address some of the limitations of current vaccines. We describe two phase 3 efficacy studies of a recombinant quadrivalent virus-like particle (QVLP) influenza vaccine manufactured in plants, one in adults aged 18-64 years (the 18-64 study) and one in older people aged 65 years and older (the 65-plus study). We did two randomised, observer-blind, multinational studies in the northern hemisphere in the 2017-18 (the 18-64 study) and 2018-19 (the 65-plus study) influenza seasons. The 18-64 study was done at 73 sites and the 65-plus study was done at 104 sites, both across Asia, Europe, and North America. In the 18-64 study, inclusion criteria were body-mass index less than 40 kg/m2; age 18-64 years at screening visit; and good health. In the 65-plus study, inclusion criteria were body-mass index of maximum 35 kg/m2; aged 65 years or older at screening visit; not living in a rehabilitation centre or care home; and no acute or evolving medical problems. Participants in the 18-64 study were randomly assigned (1:1) to receive either QVLP vaccine (30 μg haemagglutinin per strain) or placebo. Participants in the 65-plus study were randomly assigned (1:1) to receive QVLP vaccine (30 μg haemagglutinin per strain) or quadrivalent inactivated vaccine (QIV; 15 μg haemagglutinin per strain). The primary outcome in the 18-64 study was absolute vaccine efficacy to prevent laboratory-confirmed, respiratory illness caused by antigenically matched influenza strains. The primary outcome in the 65-plus study was relative vaccine efficacy to prevent laboratory-confirmed influenza-like illness caused by any influenza strain. The primary analyses were done in the per-protocol population and safety was assessed in all participants who received the assigned treatment. These studies are registered with ClinicalTrials.gov (18-64 study NCT03301051; 65-plus study NCT03739112). In the 18-64 study, between Aug 30, 2017, and Jan 15, 2018, 10 160 participants were randomly assigned to receive either QVLP vaccine (5077 participants) or placebo (5083 participants). The per-protocol population consisted of 4814 participants in the QVLP group and 4812 in the placebo group. The study did not meet its primary endpoint of 70% absolute vaccine efficacy for the QVLP vaccine (35·1% [95% CI 17·9 to 48·7]) against respiratory illness caused by matched strains. 55 (1·1%) of 5064 participants in the QVLP group versus 51 (1·0%) of 5072 in the placebo group had a serious adverse event. Four (0·1%) and six [0·1%] participants had severe treatment-related treatment-emergent adverse events. In the 65-plus study, between Sept 18, 2018, and Feb 22, 2019, 12 794 participants were randomly assigned to receive either QVLP vaccine (6396 participants) or QIV (6398 participants). The per-protocol population consisted of 5996 participants in the QVLP group and 6026 in the QIV group. The study met its primary non-inferiority endpoint with a relative vaccine efficacy of the QVLP vaccine for the prevention of influenza-like illness caused by any strain of 8·8% (-16·7 to 28·7). 263 (4·1%) of 6352 participants in the QVLP group versus 266 (4·2%) of 6366 in the QIV group had serious adverse events (one [<0·1%] vs two [<0·1%] were considered treatment-related); one (<0·1%) versus three (<0·1%) participants had severe treatment-related treatment-emergent adverse events. These efficacy studies are the first large-scale studies of any plant-derived human vaccine. Together, they show that the plant-derived QVLP vaccine can provide substantial protection against respiratory illness and influenza-like illness caused by influenza viruses in adults. QVLP vaccine was well tolerated and no major safety signal arose in participants who received QVLP vaccine across the two studies. Medicago.

A pooled genome-wide screening strategy to identify and rank influenza host restriction factors in cell-based vaccine production platforms

Cell-derived influenza vaccines provide better protection and a host of other advantages compared to the egg-derived vaccines that currently dominate the market, but their widespread use is hampered by a lack of high yield, low cost production platforms. Identification and knockout of innate immune and metabolic restriction factors within relevant host cell lines used to grow the virus could offer a means to substantially increase vaccine yield. In this paper, we describe and validate a novel genome-wide pooled CRISPR/Cas9 screening strategy that incorporates a reporter virus and a FACS selection step to identify and rank restriction factors in a given vaccine production cell line. Using the HEK-293SF cell line and A/PuertoRico/8/1934 H1N1 influenza as a model, we identify 64 putative influenza restriction factors to direct the creation of high yield knockout cell lines. In addition, gene ontology and protein complex enrichment analysis of this list of putative restriction factors offers broader insights into the primary host cell determinants of viral yield in cell-based vaccine production systems. Overall, this work will advance efforts to address the public health burden posed by influenza.

Relative Effectiveness of the Cell-Cultured Quadrivalent Influenza Vaccine Compared to Standard, Egg-derived Quadrivalent Influenza Vaccines in Preventing Influenza-like Illness in 2017-2018.

BackgroundInfluenza antigens may undergo adaptive mutations during egg-based vaccine production. In the 2017–2018 influenza season, quadrivalent, inactivated cell-derived influenza vaccine (ccIIV4) vaccine was produced using A(H3N2) seed virus propagated exclusively in cell culture, thus lacking egg adaptive changes. This United States study estimated relative vaccine effectiveness (rVE) of ccIIV4 vs egg-derived quadrivalent vaccines (egg-derived IIV4) for that season.MethodsVaccination, outcome, and covariate data were ascertained retrospectively from a electronic medical record (EMR) dataset and analyzed. The study cohort included patients ≥ 4 years of age. rVE was estimated against influenza-like illness (ILI) using diagnostic International Classification of Diseases, Ninth or Tenth Revision codes. The adjusted odds ratios used to derive rVE estimates were estimated from multivariable logistic regression models adjusted for age, sex, race/ethnicity, geographic region, and health status.ResultsOverall, 92 187 individuals had a primary care EMR record of ccIIV4 and 1 261 675 had a record of egg-derived IIV4. In the ccIIV4 group, 1705 narrowly defined ILI events occurred, and 25 645 occurred in the standard egg-derived IIV4 group. Crude rVE was 9.2% (95% confidence interval [CI], 4.6%–13.6%). When adjusted for age, sex, health status, comorbidities, and geographic region, the estimated rVE changed to 36.2% (95% CI, 26.1%–44.9%).ConclusionsccIIV4, derived from A(H3N2) seed virus propagated exclusively in cell culture, was more effective than egg-derived IIV4 in preventing ILI during the 2017–2018 influenza season. This result suggests that cell-derived influenza vaccines may have greater effectiveness than standard egg-derived vaccines.

Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells

BackgroundInfluenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms.MethodsFour H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated.ResultsThe HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses.ConclusionsOur study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.

Comparing influenza vaccine effectiveness between cell-derived and egg-derived vaccines, 2017–2018 influenza season

The Department of Defense Global Respiratory Pathogen Surveillance Program conducted a study to compare the differences in vaccine effectiveness (VE) of the cell-derived and egg-derived vaccines. DoD healthcare beneficiaries, excluding service members, that presented with influenza-like illness for the period of 1 October 2017 through 28 April 2018 were included in a test-negative case-control study examining laboratory confirmed influenza infections. Three VE analyses were performed (1) influenza infection among those vaccinated with cell-derived vaccines (2) influenza infection among those vaccinated with egg-derived vaccines and a (3) relative VE which directly compared the odds of influenza infection with cell-derived vaccine against those with egg-derived vaccines. The cell-derived and egg-derived vaccines were moderately protective against all influenza types with significant VE estimates for all dependents at 46% (95% confidence interval, 33, 56) and 53% (45, 60), respectively. Of the subtype analyses, influenza A(H1N1)pdm09 performed the best. In the cell-derived vaccine, the adult age group was moderate to high at 71% (44, 85) and children moderate at 56% (15, 75). In the egg-derived vaccine, the children age group was at a high 88% (80, 93) effectiveness and adults at 81% (56, 92). When comparing cell-derived vaccine directly to the egg-derived vaccine, the relative VE found significant results only for influenza A(H1N1)pdm09 which favored the egg-derived vaccine with odds ratios of 2.0 (1.1, 3.6) for all dependents and 2.9 (1.3, 6.3) for children. In the influenza A(H3N2) analysis, statistical significance was not gained; however, the odds favored the cell-derived vaccine.

Egg-Independent Influenza Vaccines and Vaccine Candidates.

Vaccination remains the principal way to control seasonal infections and is the most effective method of reducing influenza-associated morbidity and mortality. Since the 1940s, the main method of producing influenza vaccines has been an egg-based production process. However, in the event of a pandemic, this method has a significant limitation, as the time lag from strain isolation to final dose formulation and validation is six months. Indeed, production in eggs is a relatively slow process and production yields are both unpredictable and highly variable from strain to strain. In particular, if the next influenza pandemic were to arise from an avian influenza virus, and thus reduce the egg-laying hen population, there would be a shortage of embryonated eggs available for vaccine manufacturing. Although the production of egg-derived vaccines will continue, new technological developments have generated a cell-culture-based influenza vaccine and other more recent platforms, such as synthetic influenza vaccines.

Mechanism of a Decrease in Potency for the Recombinant Influenza A Virus Hemagglutinin H3 Antigen During Storage

The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (∼50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID.

Preflucel®: a Vero-cell culture-derived trivalent influenza vaccine

Vaccination is the principal means to reduce the impact of influenza infection. Effective vaccination programs require a reliable and safe production system. Traditionally, influenza vaccines are produced in embryonated chicken eggs. Over the last two decades, new cell culture-derived vaccines have been licensed and manufactured, and other vaccines are still in various phases of development. Vero cells have been used for the development of a wide variety of vaccines including influenza vaccines. Pandemic and avian influenza vaccines derived from Vero cells have been shown to be well tolerated and immunogenic in animal and Phase I–II clinical studies. A Phase III randomized, double-blind, placebo-controlled trial of a trivalent influenza vaccine produced in Vero-cell culture was conducted in 7250 adults aged 18–49 years. Overall protective efficacy for antigenically matched influenza vaccine was 78.5%. The vaccine was well tolerated with no treatment-related serious adverse events and compared favorably with egg-derived vaccines from previous trials. Vero-cell-derived influenza vaccines have the potential to be an important parts of the influenza vaccine strategy, especially if an avian-derived strain becomes predominant or the demand outstrips the capacity of egg-based production systems.

Clinical development of a Vero cell culture-derived seasonal influenza vaccine

BackgroundCell culture technologies have the potential to improve the robustness and flexibility of influenza vaccine supply and to substantially shorten manufacturing timelines. We investigated the safety, immunogenicity and efficacy of a Vero cell culture-derived seasonal influenza vaccine and utilized these studies to establish a serological correlate of vaccine protection. MethodsTwo multicenter, randomized, double-blind phase III trials were undertaken in the US during the 2008–2009 Northern hemisphere influenza season, in young (18–49 years) and older (50–64 years and ≥65 years) adult subjects. 7250 young adults were randomized 1:1 to receive either Vero-derived vaccine or placebo. 3210 older adult subjects were randomized 8:1 to receive either Vero-derived vaccine or a licensed egg-derived vaccine. Serum hemagglutination inhibition antibody titers were assessed 21 days post-vaccination. Vaccine efficacy in preventing cell culture-confirmed influenza infection was determined for the young adult population. Local and systemic adverse events were recorded in both studies. ResultsThe Vero-derived vaccine was safe and well tolerated in both young and older adults. All US and European immunological licensing thresholds were comfortably met in both populations. Vaccine efficacy in young adults was 79% against A/H1N1 viruses antigenically matching the corresponding vaccine strain and 78.5% for all antigenically matched influenza viruses. A hemagglutination inhibition antibody titer of ≥1:15 provided a reliable correlate of protection for the Vero-derived influenza vaccine, with no additional benefit at titers >1:30. Bridging of the correlate of protection established in the young adult population to the older adult immunogenicity data demonstrated the likely effectiveness of the Vero-derived vaccine in the older adult population. ConclusionsA Vero cell culture-derived seasonal influenza vaccine is safe, immunogenic and protects against infection with influenza virus. The novel vaccine technology has the potential to make a substantial contribution to improving influenza vaccine supply. Clinical trial registrationThe studies are registered with ClinicalTrials.gov, numbers NCT00566345 and NCT00782431.

Plaque purification as a method to mitigate the risk of adventitious-agent contamination in influenza vaccine virus seeds

At present, the seed viruses for the manufacture of licensed seasonal inactivated influenza vaccines in the United States are derived from primary egg isolates as a result of concerns associated with adventitious agents. According to the prevailing view, the passage of influenza viruses through eggs serves as a filtering step to remove potential contaminating viruses. We have investigated the feasibility of addressing adventitious-agent risk by subjecting influenza virus to a plaque-purification procedure using MDCK cells. SV40 and canine adenovirus-1 (representing viruses for which MDCK cells are non-permissive and permissive, respectively) were used as challenge viruses to model agents of concern that might be co-isolated along with the influenza virus. By mixing influenza virus strain A/PR/8/34 with varying amounts of each challenge virus and then performing a plaque assay for influenza virus using MDCK cells, we have attempted to determine the efficiency by which the challenge virus is removed. Our data suggest that substantial removal can be achieved even after a single round of plaque purification. If cell-derived isolates were deemed to be acceptable following a plaque-purification procedure, the manufacture of seasonal influenza vaccine would be facilitated by: (1) the expansion of the repertoire of viruses from which seed virus candidates could be generated for licensed egg-derived vaccines as well as for vaccines manufactured in mammalian cells; and (2) the mitigation of adventitious-agent risk associated with the seed virus, and hence the elimination of the need to passage seed viruses in eggs for vaccines manufactured in mammalian cells.

In vitro assessment of the allergenicity of a novel influenza vaccine produced in dog kidney cells in individuals with dog allergy

An inactivated influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used to immunize egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. To evaluate the allergenicity of the inactivated influenza vaccine produced in cell culture in a mediator release assay. Rat basophil leukemia (RBL) cells transfected with human IgE receptor-1 were sensitized with sera from dog-allergic adults with positive skin prick test reactions to dog extract and detectable dog dander IgE and were stimulated with serial dilutions of vaccine and dog dander extract. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation. Western blots were performed, and UniCAP was used to measure dog-specific IgE antibody levels. The median (interquartile range) level of dog dander IgE was 8.31 kU(A)/L (1.895-14.5 kU(A)/L) and of dog epithelium IgE was 3.19 kU(A)/L (0.835-6.27 kU(A)L). Median (range) maximum NHR (at the first 10-fold dilution) was 0% (0%-1.4%) to vaccine and 10.2% (0%-35.9%) to dog dander (P < .001). In an egg-allergic control subject, the maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to vaccine on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from an egg-allergic control subject strongly bound embryonated egg-derived vaccine. An influenza vaccine produced in continuous canine kidney cells did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE.

In vitro Assessment of the Allergenicity of Novel Influenza Vaccine Produced in Dog Kidney Cells in Subjects with Dog Allergy

RATIONALE: An inactivated influenza vaccine (Optaflu® is manufactured by Novartis, registered in the EU) produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used for immunizing egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. We sought to evaluate allergenicity of Optaflu in a passive sensitization assay.METHODS: RBL cells transfected with human IgE receptor-1 were sensitized with sera from adult dog-allergic subjects with positive skin prick tests to dog extract and detectable dog dander -IgE, and stimulated with serial dilutions of Optaflu and dog dander extract. N-hexosaminidase release% (NHR) was used as a marker of RBL degranulation. Western blots were performed and UniCAP was used to measure dog-specific IgE antibody levels.RESULTS: Median dog dander-IgE was 8.31 kUA/L (0.62; 67.9); median dog epithelium-IgE was 3.2 kUA/L, (0.4; 38.2). Median (range) maximum NHR (at 1st 10-fold dilution) was: 1). Optaflu- 0% (0; 1.4); 2). Dog dander-10.2% (0; 35.9), P < 0.001; and 3). Anti-IgE positive control-17.7% (1.2; 45.4). In a control egg-allergic subject, maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to Optaflu on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from a control egg-allergic subject strongly bound embryonated egg-derived vaccine.CONCLUSIONS: Optaflu, an influenza vaccine produced in continuous canine kidney cell line did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE. RATIONALE: An inactivated influenza vaccine (Optaflu® is manufactured by Novartis, registered in the EU) produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used for immunizing egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. We sought to evaluate allergenicity of Optaflu in a passive sensitization assay. METHODS: RBL cells transfected with human IgE receptor-1 were sensitized with sera from adult dog-allergic subjects with positive skin prick tests to dog extract and detectable dog dander -IgE, and stimulated with serial dilutions of Optaflu and dog dander extract. N-hexosaminidase release% (NHR) was used as a marker of RBL degranulation. Western blots were performed and UniCAP was used to measure dog-specific IgE antibody levels. RESULTS: Median dog dander-IgE was 8.31 kUA/L (0.62; 67.9); median dog epithelium-IgE was 3.2 kUA/L, (0.4; 38.2). Median (range) maximum NHR (at 1st 10-fold dilution) was: 1). Optaflu- 0% (0; 1.4); 2). Dog dander-10.2% (0; 35.9), P < 0.001; and 3). Anti-IgE positive control-17.7% (1.2; 45.4). In a control egg-allergic subject, maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to Optaflu on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from a control egg-allergic subject strongly bound embryonated egg-derived vaccine. CONCLUSIONS: Optaflu, an influenza vaccine produced in continuous canine kidney cell line did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE.

A Broadly Protective Vaccine against Globally Dispersed Clade 1 and Clade 2 H5N1 Influenza Viruses

Development of effective and immunogenic vaccines against highly pathogenic avian influenza H5N1 viruses with the potential to cause a pandemic is a public health priority. The global demand for a vaccine cannot be met in the event of an influenza pandemic because of the limited capacity to manufacture egg-derived vaccines as well as potential problems with the availability of embryonated eggs. Thus, there is an urgent need to develop alternative, egg-independent vaccines. We developed an adenoviral vector-based vaccine that contains hemagglutinin protein from clade 1 and clade 2 viruses, as well as conserved nucleoprotein, to broaden the vaccine coverage against H5N1 viruses.

New Pre-pandemic Influenza Vaccines: An Egg- and Adjuvant-independent Human Adenoviral Vector Strategy Induces Long-lasting Protective Immune Responses in Mice

Highly pathogenic avian H5N1 influenza viruses that are currently circulating in southeast Asia may acquire the potential to cause the next influenza pandemic. A number of alternate approaches are being pursued to generate cross-protective, dose-sparing, safe, and effective vaccines, as traditional vaccine approaches, i.e., embryonated egg-grown, are not immunogenic. We developed a replication-incompetent adenoviral vector-based, adjuvant- and egg-independent pandemic influenza vaccine strategy as a potential alternative to conventional egg-derived vaccines. In this paper, we address suboptimal dose and longevity of vaccine-induced protective immunity and demonstrate that a vaccine dose as little as 1 x 10(6) plaque-forming unit (PFU) is sufficient to induce protective immune responses against a highly pathogenic H5N1 virus. Furthermore, the vaccine-induced humoral and cellular immune responses and protective immunity persisted at least for a year.

Protective efficacy of intranasal cold-adapted influenza A/New Caledonia/20/99 (H1N1) vaccines comprised of egg- or cell culture-derived reassortants

Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes. However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures. To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt). Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent. When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n. with 7 × 10 10 pfu of NC/wt. In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD 50) was less than 1.2 × 10 4 pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells. Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n. vaccines.

A practical approach to immunization in atopic children.

A practical approach to immunization in atopic children.

Safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived influenza vaccine in healthy adults, seniors, and children

We performed randomized, double-blind, controlled trials to assess the safety and immunogenicity of an inactivated, Madin Darby Canine Kidney (MDCK)-derived cell line produced influenza vaccine in healthy adults (19–50 years), children (3–12 years) and the elderly (≥65 years). We studied three lots of cell culture-derived vaccine and one lot of licensed egg-derived vaccine in healthy adults ( n=462), two lots of cell culture-derived vaccine and one lot of egg-derived vaccine in seniors ( n=269), and one lot of each vaccine in children ( n=209). Adverse events were collected during the first 3 days post-immunization; serum was collected before and 1 month after immunization. Rates of local and systemic adverse reactions were similar with both vaccines. An injection site adverse event rated at least moderate severity was reported by 21.9% of children who received the egg-derived vaccine and 25.0% of those who received the cell culture-derived vaccine. In healthy adults the proportions were 12.1 and 15.3%, respectively and 6.7 and 6.3%, respectively in seniors. Systemic events of at least moderate severity were 12.4 and 12.5% in children, 19.8 and 13.6% in healthy adults, and 14.1 and 9.7% in seniors; none of these differences were statistically significant. The antibody response against all three viruses was similar between the two vaccines. From 83 to 100% of children, healthy adults and seniors achieved hemagglutination inhibition titers in excess of 40 post-immunization. We conclude that the cell culture-derived vaccine was safe and immunogenic in children, healthy adults and seniors.

Humoral and cell-mediated immunity to Vero cell-derived influenza vaccine

A candidate trivalent influenza whole virus vaccine produced in a continuous mammalian cell line (Vero), and analogous commercially available egg-derived vaccines, were compared for their ability to induce humoral and cell-mediated immunity in Balb/c mice. Substantial haemagglutination-inhibition titre and high levels of influenza virus-specific IgG were found in all groups of immunized mice, irrespective of the vaccine formulation. The IgG responses were predominantly of IgG1 and IgG2a/2b isotypes. Virus-specific secretory IgA antibodies were detected only in mice immunized intranasally with a live virus, derived either from Vero cells or eggs. T-cell proliferative responses and T-helper 1 type cytokine release was significantly higher in mice immunized with Vero cell-derived influenza vaccine compared to egg-derived vaccine formulations. We have demonstrated that the immunogenicity of the trivalent Vero cell-derived whole influenza virus vaccine was comparable to that of the equivalent egg-derived vaccine, with respect to humoral immune response and was superior with respect to cellular response.