Induction of T lymphocyte responses to dengue virus by a candidate tetravalent live attenuated dengue virus vaccine.

Development of a safe and immunogenic tetravalent dengue virus (DV) vaccine has been designated as a priority by the World Health Organization. We characterized the T cell response to DV induced by a candidate live attenuated tetravalent DV vaccine as part of a phase I study. Proliferation and cytotoxic T lymphocyte (CTL) responses to multiple DV serotypes were detected in six of six and four of four subjects studied, respectively. Proliferation responses were higher to DV serotypes 1 and 3 than to serotypes 2 and 4. CTL responses were higher to DV serotypes 2 and 3 than to serotype 1, and included serotype cross-reactive responses. Production of interferon-gamma, but not IL-4, was observed in response to DV stimulation. This candidate vaccine is immunogenic for both CD4+ and CD8+ T lymphocytes. However, T cell responses to the four DV serotypes were not equivalent, suggesting that the vaccine could be further optimized.

Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers.

A randomized, controlled, double-blinded study was conducted to determine safety and immunogenicity of five live attenuated dengue vaccines produced by Aventis Pasteur (AvP). The study was completed with 40 flavivirus non-immune volunteers: five recipients of each monovalent (dengue-1, dengue-2, dengue-3, or dengue-4) vaccine, ten recipients of tetravalent (dengue-1, dengue-2, dengue-3, and dengue-4) vaccine, and ten recipients of vaccine vehicle alone. All vaccines were administered in a single subcutaneous dose (range, 3.6-4.4 log(10) plaque forming units). No serious adverse reactions occurred in volunteers followed for 6 months after vaccination. Five vaccine recipients developed fever (T > or = 38.0 degrees C), including four tetravalent vaccinees between days 8 and 10 after vaccination. Dengue-1, dengue-2, dengue-3, or dengue-4 vaccine recipients reported similar frequency of mild symptoms of headache, malaise, and eye pain. Tetravalent vaccinees noted more moderate symptoms with onset from study days 8-11 and developed maculopapular rashes distributed over trunk and extremities. Transient neutropenia (white blood cells < 4000/mm3) was noted after vaccination but not thrombocytopenia (platelets < 100,000/mm3). All dengue-3, dengue-4, and tetravalent vaccine recipients were viremic between days 7 and 12 but viremia was rarely detected in dengue-1 or dengue-2 vaccinees. All dengue-2, dengue-3, and dengue-4, and 60% of dengue-1 vaccine recipients developed neutralizing and/or immunoglobulin M antibodies. All tetravalent vaccine recipients were viremic with dengue-3 virus and developed neutralizing antibodies to dengue-3 virus. Seven volunteers also had multivalent antibody responses, yet the highest antibody titers were against dengue-3 virus. The AvP live attenuated dengue virus vaccines are safe and tolerable in humans. The live attenuated tetravalent dengue vaccine was most reactogenic, and preferential replication of dengue-3 virus may have affected its infectivity and immunogenicity.

Experience with vero cells at Pasteur Mérieux Connaught.

The Vero cell line has been used by Pasteur-Merieux-Connaught (PMC) since 1982 with the Cell Bank's system to produce, at the 142nd passage, inactivated polio vaccine (IPV), oral polio vaccine (OPV) and rabies vaccines. The safety of the cell line has been regularly validated at the WCB level according to the WHO and European Pharmacopeia requirements for absence of bacteria, fungi, mycoplasma and viruses. Special emphasis was devoted to establishing the absence of simian viruses (SV40, SIV, Retro-D virus, simian CMV). Reverse Transcriptase (RT) activity was also negative. At low level of passage, the Vero cells are not tumorigenic. Vaccines have been prepared in low passage level Vero cells and, together with the excellent downstream purification, has resulted in excellent safety as attested by pharmacovigilance of more than 100 million doses of IPV during 12 years, more than 20 million doses of rabies vaccine during 10 years and more than one billion of OPV during six years.

[Is there an alternative to the amaril 17D vaccine?]. / Ya-t-il une alternative au vaccin amaril 17D?

The live attenuated yellow fever vaccine 17D was found as early as 1936 by M. Theiler of the Rockfeller Foundation. This strain of yellow fever is still the only one used today. The experience acquired with this vaccine has led to various changes in its composition: use of the seed lot system (in 1941) following the accidents observed in Brazil; elimination of human plasma as stabilising agent because of hepatitis B transmission (1942); preparation of a vaccine free of avian leukemia; perfection of a thermostable vaccine (1984). These various successive improvements resulted in one of the most effective vaccines. Over the past years, different ways of improving the vaccine have been envisaged: change of cellular substrate, purifications, development of a new vaccine through genetical engineering. We will review these different approaches in order to gauge their advantages and drawbacks both from a legislative and pharmaceutical point of view. It has been recently suggested that an infected cDNA clone from the 17D strain be used as a yellow fever vaccine or as a gene-vector for other flaviviruses. This most promising approach raises questions, notably ones of security and legislation which we will discuss.

Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment.

The 74HB59 strain of Rift Valley fever (RVF) virus, isolated from a human case in the Central African Republic, was shown to be composed of a heterogeneous population of viruses when plaque-purified clones were analyzed for their reactivity with monoclonal antibodies (MAbs) directed against the nucleocapsid (N) protein or the nonstructural (NSs) protein. One of these clones, C13, was of particular interest in that it proved to be avirulent in mice and hamsters, and highly immunogenic. Although C13 showed normal reactivity with a large panel of MAbs directed at the glycoproteins, it failed to react with specific MAbs or polyclonal antibodies directed at the NSs protein and with a specific MAb recognizing the N protein of the Egyptian strains. Consequently, the small RNA segment, which encodes the N and NSs proteins in an ambisense strategy, was sequenced and compared with the existing sequence of the attenuated MP-12 RVF virus strain. We found that the NSs gene contained, in addition to two conservative coding changes, a large internal deletion of 549 nucleotides that removes 69% of the open reading frame but conserves in-frame the N and C termini of the predicted translation product. In addition, the sequence revealed that the N protein of C13 contained a single amino acid change. Clone C13 replicated normally in certain cell types in vitro and in Culex pipiens mosquitoes after intrathoracic inoculation, but established abortive infections in MRC-5 human fibroblasts.

Short report: isolation and partial characterization of a Puumala virus from a human case of nephropathia epidemica in France.

Puumala (PUU) virus (Bunyaviridae: Hantavirus), the etiologic agent of nephropathia epidemica (NE), the mid form of hemorrhagic fever with renal syndrome, is enzootic in Europe and has been known to occur in France since 1983. We report the first isolation of PUU virus in France and western Europe from a case of NE acquired in France. The virus was isolated from a serum collected in the acute phase of the clinical course by successive blind passages in Vero E6 cells. Serologic typing using monoclonal antibodies confirmed the identity of the virus as PUU. The sequence of an 832-nucleotide fragment of the virus medium RNA segment obtained by the polymerase chain reaction (PCR) also classified it as a PUU virus. The sequence of this isolate from a human case in France is closely related to the sequence of a PUU virus obtained by the PCR from a German patient.

Antigenic comparison of Ockelbo virus isolates from Sweden and Russia with Sindbis virus isolates from Europe, Africa, and Australia: further evidence for variation among alphaviruses.

The plaque-reduction neutralization test (PRNT) was used to compare 15 isolates of Ockelbo virus from Sweden, one isolate of Ockelbo virus from Russia, the Egyptian prototype Sindbis virus, and Sindbis-like viruses from Slovakia, South Africa, Cameroon, and Australia. Strains from northern Europe (Sweden and Russia) were indistinguishable by PRNT. We observed some antigenic variation between isolates of Sindbis virus from Europe, Africa, and Australia. An Australian strain (C-377) was shown to be distinct from prototype Sindbis virus, and the Acrocephalus strain from Slovakia was shown to be identical to prototype Sindbis virus. All other strains, including Ockelbo virus isolates, were shown to be subtypes of prototype Sindbis virus.

Rift Valley fever on the east coast of Madagascar.

In March 1990, a Rift Valley fever virus (RVFV) outbreak was suspected in the district of Fenerive on the east coast of Madagascar after an abnormally high incidence of abortions and disease in livestock. Sera from humans and cattle were tested for RVFV antibodies by immunofluorescence assay (IFA) and ELISA-IgM capture. Sera and mosquitoes collected in the same area were tested for virus isolation by tissue culture and suckling mouse intracerebral inoculation, and for antigen detection by an ELISA antigen capture assay. Among cattle from the area, RVFV antibody prevalence was 58.6% by IFA and 29.6% by ELISA-IgM. In contrast, human populations in the same area had a lower RVFV antibody prevalence, with 8.01% IFA and 5.4% IgM-positive sera. No RVFV antigen was detected and virus isolation was unsuccessful from the sera and mosquito pools tested. Different hypotheses concerning the emergence and diffusion of RVFV in this area and the occurrence of the outbreak are discussed.

[Epidemiology of Rift Valley fever in west Africa. 1. Serological investigation of small ruminants in Niger]. / Epidémiologie de la fièvre de la Vallée du Rift en Afrique de l'ouest. 2. Enquête sérologique chez les petits ruminants au Niger.

A serosurvey of Rift Valley Fever virus infection conducted among 557 sheep and 643 goats from Niger in 1986 points out that 2.8% of the 1,200 animals tested had RVF virus reacting antibodies. The circulation of the virus is demonstrated, as well for another phlebovirus related to RVF virus, the strain Arumowot.

Generation and transmission of Rift Valley fever viral reassortants by the mosquito Culex pipiens.

Reassortant viruses containing heterologous S and M genomic RNA segments were obtained from both mosquito and vertebrate hosts that had been coinfected with Egyptian and Senegalese strains of Rift Valley fever (RVF) virus. The origin of the S and M RNA segments in each plaque-cloned virus was determined with monoclonal antibodies capable of differentiating the nucleocapsid protein (S segment marker) or the G1 glycoprotein (M segment marker) of the parental strains. In the mosquito Culex pipiens, reassortants were detected after sequential ingestion of parental viruses by interrupted feeding on two infected hamster hosts, after feeding on a single host that had been infected with both parental strains, and from individual mosquitoes inoculated intrathoracically with both parental strains. Reassortant viruses replicated efficiently in mosquitoes and were readily transmissible by bite to hamsters. Replication of a second infecting strain of RVF virus was, however, completely inhibited if that virus was inoculated into a mosquito greater than or equal to 48 h after the first viral strain. Genetic reassortment may provide a mechanism for increased heterogeneity, and thus affect the epidemiology and evolution of RVF virus.

Use of reassortant viruses to map attenuating and temperature-sensitive mutations of the Rift Valley fever virus MP-12 vaccine.

A live-attenuated vaccine for Rift Valley fever virus (RVFV), MP-12, has been developed recently by undirected, serial mutagenesis of a RVFV strain (ZH548) isolated during the 1977 epidemic in Egypt. In the present study, the mutations responsible for attenuation of this virus have been examined by analysis of reassortant viruses generated between the vaccine strain and a wild RVFV strain isolated in Senegal. Reassortant viruses were generated efficiently in multiply infected Vero cells, and were readily isolated without application of selective pressures. The origin of the S and M genomic RNA segments in each cloned reassortant virus was determined with monoclonal antibodies capable of differentiating the nucleocapsid protein (S segment marker) or G1 glycoprotein (M segment marker) of the parental strains. The L segment of the vaccine strain was found to contain a temperature-sensitive (ts) mutation, and the origin of the L segment in most reassortants could be inferred by analysis of their ts phenotype. Analysis of the virulence properties of selected reassortant viruses in mice demonstrated that virulence characteristics were under polygenic control, and that at least one mutation capable of independently attenuating the virus existed on each genome segment. The L and M RNA segments were also found to contain ts mutations. These findings suggest that reversion to virulence is unlikely, and further indicate that genetic reassortment with wild-type viruses during a vaccination programme in endemic areas would also be expected to yield attenuated variants.

Comparison of in vitro and in vivo systems for propagation of Rift Valley fever virus from clinical specimens.

Several cell cultures and animals were compared for their relative sensitivity as primary isolation systems for Rift Valley fever virus (RVFV) and to determine if virulence characteristics of the isolates were altered in these systems. Eleven human sera from known cases of Rift Valley fever (RVF) were obtained from the 1987 epidemic in Mauritania and served as the source of virus for these studies. Sera were inoculated directly into cell cultures (Vero, C6/36 and DBS-FRhL-2) and animals (ICR suckling mice, Lak:LVG(SYR) hamsters and WF rats) concurrently. The cell lines provided a quick method to propagate, quantitate and identify these specimens without prior adaption. The isolates were highly virulent for suckling mice and hamsters, but not for WF rats, even after cell culture passage, which indicated that the Mauritanian isolates more closely resembled those strains from sub-Saharan Africa than those from the 1977-78 Egyptian epidemic.

Antigenic and biological properties of Rift Valley fever virus isolated during the 1987 Mauritanian epidemic.

The antigenic and biological properties of three strains of Rift Valley fever virus (RVFV) isolated during the 1987 epidemic in Mauritania were compared with those of strains isolated previously in West Africa and with other selected African strains. Neither the antigenic characteristics of the Mauritanian isolates, as monitored by the binding of 59 monoclonal antibodies, nor the electrophoretic migration of the virus-specific structural and non-structural proteins were significantly different from other strains of RVFV isolated in this region or elsewhere in sub-Saharan Africa. Biological and antigenic traits which distinguished the strains isolated from the 1977 Egyptian epidemic were not associated with the Mauritanian isolates.

Use of heat inactivated viral haemorrhagic fever antigens in serological assays.

Heating for 1 h at 60 degrees C completely destroyed the infectivity of sucrose-acetone-extracted antigen of Rift Valley (RVF) and Congo Crimean haemorrhagic fever (CCHF), as well as of RVF- and CCHF-infected mouse brain. These antigens could be successfully used, however, for complement fixation and IgM-capturing enzyme immunoassay. Vero E6 cell suspensions infected with hantaviruses such as Hantaan 76-118, Tchoupitoulas, SR 11, GB-B, CG 18-20, Hällnäs, CG 13891, Seoul and Prospect Hill, as well as Vero cells infected with CCHF and RVF viruses, were completely inactivated after heating for 1 h at 60 degrees C. Indirect immunofluorescent antibody test results obtained on slides prepared with heat-inactivated cell suspensions correlated well with results obtained on slides prepared with unheated cell suspensions. Inactivation is a simple, rapid, economic and reproducible method for inactivation of hantaviruses and CCHF and RVF viruses, with preservation of the ability to react specifically with antibodies.

Rapid diagnosis of human Crimean-Congo hemorrhagic fever and detection of the virus in naturally infected ticks.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect Crimean-Congo hemorrhagic fever (CCHF) virus-specific immunoglobulin M (IgM) in human serum samples. For this test, a heat-inactivated antigen was prepared from the brains of suckling mice infected with CCHF virus. The IgM-capture ELISA proved more sensitive than indirect fluorescence tests for IgM to this virus. A human serum containing high-titer IgM to CCHF virus was used for an antigen-capture ELISA to detect this virus in heat-inactivated suspensions of virus-infected ticks. The antigen-capture ELISA appeared to be as sensitive as virus isolation in suckling mice. The studies described suggest that the IgM-capture ELISA and the antigen-detection ELISA should provide a rapid and sensitive diagnosis of human CCHF virus infection and should be useful in ecologic studies of this virus.