Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen.

     

Attempts to vaccinate ewes and their lambs against natural infection with Haemonchus contortus in a tropical environment

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50 μg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3 week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1 month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50 μg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus.     

Challenge exposure of sheep immunized with live vaccine and culture supernatant of Mannheimia haemolytica A1: Effects of revaccination

Vaccination against Mannheimia haemolytica is a routine procedure in sheep farming, being repeatedly performed on the recommendations of commercial veterinary manufacturers or due to outbreaks of respiratory disease. However, the consequences of repeat vaccination after a short period, using live vaccines or culture supernatant (CS), have not been established, and its benefits are unknown. This study aims to evaluate, in a challenge exposure, the effects of revaccinating sheep with this type of biological product. Thus, 20 mixed-breed, 6–9-month-old sheep were divided into four groups, with five animals in each. Animals in group I were inoculated subcutaneously with 2 ml of a 1 × 10⁹ CFU/ml suspension of bacteria in the exponential growth phase. Group II was inoculated with the same bacterial concentration but in the stationary phase. Animals in group III were inoculated with 3 ml of CS and group IV with saline solution, as control. Each group was immunized again 14 days later, following the same guidelines. On day 21, they were intranasally exposed to parainfluenza-3 virus (PI-3). On day 27, they were challenged with a transthoracic injection of live M. haemolytica (2 × 10⁹ CFU/ml). In this experiment, the levels of agglutinating and antileukotoxin antibodies were evaluated through indirect hemagglutination and toxin neutralization assays. During the first 24 h post-challenge, four animals in group I died, three in group II and two in group III, even though they had developed significant levels of agglutinating and antileukotoxin antibodies. Only one animal from the control group died. Thus, contrary to the development of improved protection due to continuous immunizations with live bacteria or CS, a negative effect was observed. This can be explained as the result of a hypersensitivity type III reaction, due to the significant decrease in agglutinating antibodies observed shortly after the challenge, indicating the formation of immune complex, which triggered the release of chemical mediators of inflammation that, finally, promoted edema and pulmonary congestion.     

Increased extracellular pressure provides a novel adjuvant stimulus for enhancement of conventional dendritic cell maturation strategies

Dendritic cell (DC)-based vaccine strategies have gained increasing popularity in recent years. Methods for ex vivo generation of immunocompetent mature DCs still require optimization. DCs have been shown to phenotypically mature under elevated pressure. We compared the effects of pressure on DC maturation with LPS- and cytokine-stimulation. Human monocyte-derived immature or LPS- and cytokine-matured DCs were exposed to ambient or 40 mmHg increased pressure for 12 h, then assessed for expression of CD80, CD86, CD40, MHC-I/II, and inflammatory cytokine production. DCs were also evaluated for capacity to stimulate T-cell proliferation by co-culture with allogeneic lymphocytes. Pressure significantly increased cytokine production and expression of all surface molecules on immature DC other than MHC-I and CD40. Pressure/LPS-treated DCs displayed further upregulation of MHC-I, CD40, and IL-12p70. Cytokine-matured DCs appeared less responsive to pressure. T-cell proliferation correlated with MHC expression. Results suggest mechanical stimulation of DCs may provide a useful adjuvant to TLR-agonist maturation strategies.     

Vaccines against protozoal diseases of veterinary importance

Abstract Protozoan parasites are important animal and human pathogens. At present, most of these infections are controlled by chemotherapy. In addition, vaccines are available for some of these diseases. There is, however, still an urgent need for the development of vaccines against protozoal diseases, since the current array of available vaccines is very limited. This review describes the different approaches that have been taken to develop such vaccines and discusses the difficulties that hampered vaccine development. Many of the problems are related to the complex life cycle of these parasites and the virtual lack of mass in vitro culture systems. We also give an overview of the commercial and non-commercial vaccines that do exist at present. Finally, we describe the future directions of this interesting field. New techniques and strategies include parasite cultivation methods and recombinant-DNA techniques, such as vector vaccines and DNA-vaccines. Moreover, these approaches are complemented by the development of sophisticated adjuvants; the coupling of immunoprotective molecules to entities with adjuvant activity or the use of cytokines, e.g. IL-12. Through these innovations new vaccines against protozoal diseases will become available in the near future.     

Feline immunodeficiency virus vaccine: Implications for diagnostic testing and disease management

Feline immunodeficiency virus (FIV) is a common feline pathogen, with an overall infection prevalence of approximately 11% in cats worldwide. Most infected cats eventually succumb due to direct viral effects or, more commonly, to secondary infections resulting from virus-induced immunosuppression. FIV infection is considered lifelong, and diagnosis most often relies on detection of virus-specific antibodies. A currently available whole virus, adjuvanted, inactivated FIV vaccine induces antibodies in vaccinates that is indistinguishable from those induced by infection. As a result, currently available diagnostic tests cannot reliably distinguish vaccinated cats from infected cats, or from cats that are both vaccinated and infected. From both an epidemiologic and an individual cat perspective, it is impossible to determine whether use of this vaccination is more beneficial than it is harmful.     

Prevalence of Met-203 type spaA variant in Erysipelothrix rhusiopathiae isolates and the efficacy of swine erysipelas vaccines in Japan

Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile-257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD₅₀ values of 0.45–1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan.     

Dendritic cells in Leishmania infection

Dendritic cells (DCs) are key elements of the immune system, which function as sentinel in the periphery and alert T lymphocytes about the type of invading antigen and address their polarisation, in order to mount an efficacious immune response. Leishmania spp. are parasitic protozoa which may cause severe disease in humans and domestic animals. In this work, the main studies concerning the role of DCs in Leishmania infection are reviewed, in both the murine and human models. In particular, the importance of the genetic status of the hosts and of the different Leishmania species in modulating DC-mediated immune response is examined. In addition, different approaches of DC-based vaccination against experimental leishmaniasis, which could have important future applications, are summarised.     

浅谈WHO国家疫苗监管体系评估及其上市许可板块要求

通过卫生组织国家疫苗监管体系(National Regulatory Authority)评估是一个国家疫苗具备参与联合国全球采购资格的必要条件。本文对WHO国家疫苗监管体系评估整体加以简述,重点对上市许可板块指标进行归纳,通过探讨WHO国家疫苗监管体系评估上市许可部分的管理理念,以期为疫苗监管相关部门加强自身管理提升监管质量和效率有所提示。