Evaluation of the safety, reactogenicity and immunogenicity of FluBlok® trivalent recombinant baculovirus-expressed hemagglutinin influenza vaccine administered intramuscularly to healthy adults 50-64 years of age.

BACKGROUND: Alternative methods for influenza vaccine production are needed to ensure adequate supplies. METHODS: Healthy adults 50-64 years were assigned randomly to receive one intramuscular injection of trivalent recombinant hemagglutinin (rHA) or U.S. licensed trivalent inactivated vaccine (TIV) containing H1, H3 and B antigens (Ag) derived from 2007 to 2008 influenza virus strains A/Solomon Islands/03/2006 (H1N1), A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004. Each rHA dose contained 45 µg HA/strain of the 2007-2008 FDA-recommended Ag vs. 15 µg/strain for TIV. Antibody (Ab) responses were measured using a hemagglutination-inhibition (HAI) assay at baseline and 28 days post-vaccination. Respiratory samples for viral culture were collected from subjects with influenza-like illness (ILI) during the 2007-2008 season in the U.S. RESULTS: 601 subjects were enrolled. Vaccines were well tolerated. Seroconversion (the percentage of subjects with either (a) a pre-vaccination HAI titer ≤ 10 and a post-vaccination HAI titer ≥ 40 or (b) a pre-vaccination titer ≥ 10 and a minimum four-fold rise in post-vaccination HAI antibody titer) in the TIV and rHA groups, respectively, was obtained in 66% vs. 72% for H1; 44% vs. 61% for H3; and 41% vs. 41% for B. Proportions achieving titers ≥ 40 were 96% vs. 96% for H1, 75% vs. 85% for H3, and 94% vs. 93% vs. B. Geometric mean titer ratios at day 28 (TIV/rHA) were 0.77 for H1; 0.58 for H3; and 1.05 for B, respectively. ILI frequencies were low and similar in both groups. CONCLUSIONS: Both vaccines were safe and immunogenic. Ab responses vs. H1 and H3 Ags were significantly higher in the rHA group, with similar responses to B. Furthermore, the FluBlok group had a statistically significantly higher seroconversion rate against influenza A/H3N2 compared to the TIV group.

Human papillomavirus and HPV vaccines: a review.

Cervical cancer, the most common cancer affecting women in developing countries, is caused by persistent infection with "high-risk" genotypes of human papillomaviruses (HPV). The most common oncogenic HPV genotypes are 16 and 18, causing approximately 70% of all cervical cancers. Types 6 and 11 do not contribute to the incidence of high-grade dysplasias (precancerous lesions) or cervical cancer, but do cause laryngeal papillomas and most genital warts. HPV is highly transmissible, with peak incidence soon after the onset of sexual activity. A quadrivalent (types 6, 11, 16 and 18) HPV vaccine has recently been licensed in several countries following the determination that it has an acceptable benefit/risk profile. In large phase III trials, the vaccine prevented 100% of moderate and severe precancerous cervical lesions associated with types 16 or 18 among women with no previous infection with these types. A bivalent (types 16 and 18) vaccine has also undergone extensive evaluation and been licensed in at least one country. Both vaccines are prepared from non-infectious, DNA-free virus-like particles produced by recombinant technology and combined with an adjuvant. With three doses administered, they induce high levels of serum antibodies in virtually all vaccinated individuals. In women who have no evidence of past or current infection with the HPV genotypes in the vaccine, both vaccines show > 90% protection against persistent HPV infection for up to 5 years after vaccination, which is the longest reported follow-up so far. Vaccinating at an age before females are exposed to HPV would have the greatest impact. Since HPV vaccines do not eliminate the risk of cervical cancer, cervical screening will still be required to minimize cancer incidence. Tiered pricing for HPV vaccines, innovative financing mechanisms and multidisciplinary partnerships will be essential in order for the vaccines to reach populations in greatest need.

Lessons from preventive infectious disease vaccines.

Preventive vaccines for infectious disease indications are regulated by the Office of Vaccines Research and Review, CBER, FDA. FDA-regulated biological products, including vaccines, must be safe, pure, potent, and manufactured consistently according to current Good Manufacturing Practices. Regulatory issues include common principles of vaccine production and quality control, lot release testing, vaccine preclinical data, and clinical development. For Phase 3 studies, the following should be addressed: the case definition of the disease that the vaccine is intended to prevent (piloted during Phase 2) and the sample size to support efficacy and safety considerations. Also during development, validation data for immunological and other critical assays, clinical lot consistency studies, and the possible need for bridging studies, e.g., to support manufacturing changes or extrapolation to different populations, should be addressed.

Prelicensure evaluation of combination vaccines.

There is considerable public health interest in licensing safe and effective combination vaccines. Because combination vaccines may progress rapidly from phase 1 to a pivotal phase 2 immunogenicity trial, a rigorous approach to address product issues early in development is warranted. Clinical studies to evaluate the safety, immunogenicity, and (when necessary) clinical end point efficacy of combination vaccines should be randomized and well controlled in most cases. A large phase 3 safety study (i.e., a study that enrolls thousands of vaccinees) should be included in the development plan if a phase 3 (clinical end point) efficacy trial will not be conducted. Often, the new combination vaccine under development contains immunogens that have all been previously licensed, have demonstrated efficacy in earlier clinical trials, or both. For such products, comparative immunogenicity data may be sufficient to support efficacy. When applicable, clinical data to support simultaneous administration with other relevant vaccines should be obtained. Given the complexity of combination vaccine development, early consultation with United States Food and Drug Administration can be invaluable.

Developing new smallpox vaccines.

New stockpiles of smallpox vaccine are required as a contingency for protecting civilian and military personnel against deliberate dissemination of smallpox virus by terrorists or unfriendly governments. The smallpox vaccine in the current stockpile consists of a live animal poxvirus (Vaccinia virus [VACV]) that was grown on the skin of calves. Because of potential issues with controlling this earlier manufacturing process, which included scraping VACV lesions from calfskin, new vaccines are being developed and manufactured by using viral propagation on well-characterized cell substrates. We describe, from a regulatory perspective, the various strains of VACV, the adverse events associated with calf lymph-propagated smallpox vaccine, the issues regarding selection and use of cell substrates for vaccine production, and the issues involved in demonstrating evidence of safety and efficacy.

Intent-to-treat analysis and preventive vaccine efficacy.

While the intent-to-treat (ITT) concept has often been discussed and debated in the literature with respect to randomized, placebo-controlled therapeutic trials, there has been little discussion of this issue in the context of preventive vaccine efficacy trials. ITT analysis has traditionally played a minor role (if any) in the latter trials. This paper discusses the ITT approach to analysis in randomized superiority trials of preventive vaccine efficacy, using clinical endpoints. Data are presented from published literature as well as from a simple mathematical model. The data suggest that when compliance and efficacy are high, both ITT and "per-protocol" approaches generally lead to similar conclusions regarding the acceptability of a vaccine for use in a population. However, when compliance is low, the ITT and per-protocol estimates of vaccine efficacy can be widely disparate. ITT and per-protocol analyses address unique and relevant scientific questions, and often both will be informative in evaluating preventive vaccines.

Recombinant bacillus Calmette-Guérin as a potential vector for preventive HIV type 1 vaccines.

In August 1997, the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) convened an expert working group to discuss current strategies for the development of HIV type 1 vaccines. Based on the recent findings of investigators from Japan's National Institute of Infectious Diseases (NIID) in Tokyo using recombinant bacillus Calmette-Guérin (rBCG) as a potential vectored vaccine for HIV, a recommendation was made that further work in this area is a priority. As a result, the working group reconvened in September 1998 to discuss the progress to date with this vaccine approach, as well as areas of related research to assess the feasibility of a BCG-vectored HIV vaccine. This report summarizes the discussions addressing the available scientific data on the potential use of rBCG as a vector for preventive HIV vaccines, the work necessary to move such candidate vaccines into Phase 1 clinical trials, and recommendations targeted at facilitating the long-term development of rBCG-vectored HIV vaccines.

Clinical safety evaluation of combination vaccines.

The development plan for new combination vaccines should include clinical studies designed to provide an adequate safety database (as well as efficacy data) to support licensure. Ideally, randomized studies to compare the safety of the combination vaccine with the separately administered components (or already licensed combinations of components) should be performed. For new combination vaccines having components with proven efficacy, comparative immunogenicity data may provide a sufficient basis to support efficacy, precluding the need for a large efficacy study. In the absence of the safety database that would have been derived from such an efficacy study, it is important to conduct a comparative safety study with a sample size suitable for the evaluation of less common adverse events. For large safety trials, a simplified design where only a subset of subjects is assessed in detail for the more common adverse events may be appropriate.

HIV vaccine development and evaluation: realistic expectations.

As of January 1998, more than 85 vaccines for 24 clinical indications are currently licensed in the United States. From the time of discovery of the etiologic agent to the development of a licensed vaccine, many years have usually been required. Although many vaccines have been licensed on the basis of one efficacy trial, multiple vaccine concepts and multiple efficacy trials (both in the United States and internationally) have at times been necessary. Over a relatively short period of time, there has been remarkable progress in human immunodeficiency virus (HIV) vaccine development, with over 34 different HIV candidate vaccines having been tested in phase 1 trials, and three having been tested in phase 2 trials. In spite of our incomplete understanding of HIV pathogenesis and correlates of protection, the first phase 3 efficacy trial has been initiated in the U.S. and tentative plans have been announced for three other phase 3 efficacy trials with the most advanced HIV candidate vaccines to begin in the next 3 years. Like many previous vaccine development efforts, these initial HIV vaccine efficacy trials could be the first of many large-scale efficacy trials in the future, testing various vaccine design concepts among different high-risk populations in both developed and developing countries. The choice of when and how to proceed to phase 3 trials remains a complex decision, but it is likely that only through such trials will further knowledge be gained to advance this important effort and reach our goal of a safe and effective HIV vaccine.

Preventive HIV type 1 vaccine clinical trials: a regulatory perspective.

The clinical development of a human immunodeficiency virus (HIV) vaccine should be planned so that adequate safety and efficacy data can be obtained in an efficient manner to permit a risk/benefit assessment. Phase 1 and 2 studies should support the selection of an appropriate vaccine formulation, dose and schedule for evaluation in efficacy trials. Evaluation of the immune response(s) elicited by an HIV vaccine has an important role, even early in clinical development. Immune assay results and viral detection/quantitation technology may be used to identify and characterize HIV infections occurring during the trials. Thus, information about the performance parameters of these assays is important. Considerable research and development may be needed in this regard, especially when multiple endemic HIV-1 subtypes (clades) are expected. Prior to initiating an efficacy trial, background epidemiological information (e.g., recent seroincidence, endemic clades), as well as safety and immunogenicity data with the candidate vaccine, should be obtained in the intended efficacy trial population. The effects of antiretroviral therapy use and sensitive viral detection assays on the evaluation of the primary efficacy end point (as well as secondary end points) are important considerations. The detailed statistical plan for an efficacy trial should consider the 95% confidence limits on the estimate of vaccine efficacy; this may be of exceptional importance when relatively low point estimates of efficacy are expected.

Traditional approach preventive HIV vaccines: What are the cell substrate and inactivation issues?

A workshop was convened to discuss safety issues for traditional-approach HIV vaccines, especially inactivated vaccines. The topics included issues pertaining to (1) cell substrates used for production and (2) vaccine virus inactivation. The use of cell substrates such as tumor-derived continuous cell lines (TCLs) or virus-transformed. CLs may be the most feasible approach to provide commercial-scale virus yields. However, especially because of concerns about tumorigenicity, TCLs have not been used to produce preventive vaccines for human trials with healthy subjects in the United States. Residual TCL material (e.g., DNA, cellular proteins, viruses) may not be removed during purification of intact HIV virions to the same extent achievable for a recombinant protein. Manufacturing processes, e.g., physicochemical methods of destroying DNA, could decrease tumorigenicity risk. Methods to assess potential for tumorigenicity may need further development. Another potential substrate for viral production that merits further study is human peripheral blood mononuclear cells (PBMCs). Regardless of the cell substrate used, extensive testing for adventitious agents (including non-HIV retroviruses) is needed. Vaccine virus inactivation can be considered in statistical terms, i.e., the probability of a surviving infectious particle. One formula to determine a "safety margin" (SM) is reduction of titer in log10 for all inactivation steps minus initial viral infectivity in log10. Calculations for appropriate SMs should include all sources of variability (e.g., lot-to-lot differences). Ensuring a specified SM, as the lower bound of the 95% confidence interval, for production lots was discussed. Sensitivity and specificity of infectivity assays may present limitations.

Selected regulatory and scientific topics for candidate rotavirus vaccine development.

Various aspects of the development of rotavirus vaccine candidates are discussed. As is true with other vaccines, comprehensive testing must be done to detect the possible presence of adventitious agents in the vaccine and seed preparations. Consideration must also be given to other biologic materials that come in contact with the vaccine preparation during production to prevent the introduction of contaminants. The clinical testing of rotavirus vaccines from early safety and immunogenicity studies through final efficacy studies is also discussed. Issues surrounding coadministration of investigational rotavirus vaccines with US-licensed vaccines are ideally addressed before initiation of efficacy trials. Other subjects discussed are identification of correlates of protection, multivalent vaccines, foreign efficacy trials, safety data, and statistical considerations. Sponsors of investigational vaccines are urged to contact the Food and Drug Administration for guidance during the development process, especially before the investigational new drug application and pivotal efficacy trial stages.

Public health implications of emerging vaccine technologies.

The field of public health and medicine stands to benefit immensely from the emerging vaccine technologies and improved application of existing technologies. Technological advances may promote: (1) greater flexibility and simplicity in the design and operation of immunization campaigns or ongoing prevention programs, including reduction in number of vaccine doses, cold chain elimination, slow-release/prolonged antigenic stimulation, reduced cost and hazard and increased ease of administration through noninvasive, oral delivery systems, greater population levels of immunization and health; (2) the development of documents by FDA, WHO, and other regulatory authorities and groups, to assist the manufacturer in the appropriate manufacturing, preclinical, and clinical development of these new vaccines; (3) a greater array of vaccines to protect the civilian and military populations; (4) increased vaccine potency; (5) vaccines eliciting mucosal immunity, cytotoxic T cells, and/or neutralizing antibody. At the end of the 20th century there remain many unconquered pathogens and noninfectious indications for which medical science suggests that vaccines could be effective. New technologies may provide the best hope to address this wide array of public health needs.

Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1).

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.

Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry.

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.

Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells.

Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.

Normal sister chromatid exchange frequencies during growth of a transplantable murine myeloid leukemia.

Frequencies of sister chromatid exchange (SCE) were analyzed in normal and coexisting leukemic cells harvested from the bone marrow of mice 10, 15, 18, and 21 days after transplantation of myeloid leukemic cells. These posttransplantation stages correspond to no abnormal physical or clinical symptoms (day 10) through the terminal stage of leukemia (day 21). The data indicate that the SCE frequencies in normal cells of leukemic mice did not differ from those in normal cells of normal mice. Furthermore, the frequencies in the coexisting normal and leukemic cells remained statistically constant throughout the posttransplantation period. It is concluded from this study that spontaneous cellular SCE frequencies may not be altered by the presence or growth of leukemic cells.