Low pathogenic avian influenza A(H7N9) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies.

Infections with low pathogenic avian influenza (LPAI) A(H7N9) viruses have caused more than 100 hospitalized human cases of severe influenza in China since February 2013 with a case fatality rate exceeding 25%. Most of these human infections presented with severe viral pneumonia, while limited information is available currently on the occurrence of mild and subclinical cases. In the present study, a ferret model for this virus infection in humans is presented to evaluate the pathogenesis of the infection in a mammalian host, as ferrets have been shown to mimic the pathogenesis of human infection with influenza viruses most closely. Ferrets were inoculated intratracheally with increasing doses (>10 e5 TCID50) of H7N9 influenza virus A/Anhui/1/2013 and were monitored for clinical and virological parameters up to four days post infection. Virus replication was detected in the upper and lower respiratory tracts while animals developed fatal viral pneumonia. This study illustrates the high pathogenicity of LPAI-H7N9 virus for mammals. Furthermore, the intratracheal inoculation route in ferrets proofs to offer a solid model for LPAI-H7N9 virus induced pneumonia in humans. This model will facilitate the development and assessment of clinical intervention strategies for LPAI-H7N9 virus infection in humans, such as preventive vaccination and the use of antivirals.

Modification of the ferret model for pneumonia from seasonal human influenza A virus infection.

The primary complication of seasonal influenza in humans is viral pneumonia. A conventional animal model--intranasal inoculation of ferrets with 10(6) median tissue culture infectious dose of virus--results in disease that is neither consistent nor comparable with severe viral pneumonia in humans. Therefore, the authors modified the experimental procedures by increasing the median tissue culture infectious dose to 10(9) and by inoculating via the intratracheal route, testing these procedures with H1N1 strains (A/Bilthoven/3075/1978 and A/Netherlands/26/2007) and H3N2 strains (A/Bilthoven/16190/1968 and A/Netherlands/177/2008) of seasonal influenza virus. The ferrets of all groups (n = 3 per virus strain) had clinical signs, increased body temperature, virus excretion from day 1, loss of body weight, and increased relative lung weight at 4 days postinoculation. All ferrets had severe pulmonary consolidation, and histologic examination revealed moderate to severe necrotizing bronchointerstitial pneumonia with severe edema, necrosis of alveolar epithelium, inflammatory infiltrates in alveolar septa and lumina, epithelial regeneration, and perivascular and peribronchiolar inflammatory infiltrates. The lesions were associated with the presence of influenza virus antigen in respiratory epithelium by immunohistochemistry. Although all 4 virus strains caused pulmonary lesions of comparable severity, virus isolation in the lungs, trachea, nasal concha, and tonsils showed higher mean virus titers in the H1/07 and H3/68 groups than in the H1/78 and H3/08 groups. In conclusion, the above H1N1 and H3N2 strains cause severe pneumonia in ferrets by use of the modified experimental procedures and provide a good model for pneumonia caused by seasonal influenza A virus infection in humans.

Feasibility of single-shot H5N1 influenza vaccine in ferrets, macaques and rabbits.

The feasibility of a single-shot, low-dose vaccination against pandemic influenza was investigated. The immunogenicity and safety of whole inactivated, cell culture-derived H5N1 virus plus CoVaccine HT™ as adjuvant was tested in various animal species. In ferrets, doses of 4.0 and 7.5 µg H5N1 (NIBRG-14; A/Vietnam/1194/04; clade 1) without adjuvant gave low geometric mean haemagglutination inhibition (HI) titres (GMTs) of 21-65 three weeks after intramuscular (IM) injection. The addition of 0.25-4 mg CoVaccine HT™ resulted in GMTs of 255-1470 corresponding with 4-25-fold increases. A second immunization caused GMTs of 8914-23,525 two weeks later, which confirmed strong priming. One out of 8 ferrets injected with antigen alone and 5 out of 32 ferrets injected with adjuvanted H5N1 demonstrated minimal transient, local reactions and two animals immunized with adjuvanted H5N1 exhibited increased body temperature one day after injection. In macaques, 5 µg H5N1 with CoVaccine HT™ or aluminium hydroxide as adjuvant elicited GMTs of 172 and 11, respectively three weeks later. A second immunization resulted in GMTs of 1751 and 123, respectively four weeks later. Analysis of cross-reactivity of antibodies after the first immunization with NIBRG-14 adjuvanted plus CoVaccine HT™ revealed GMTs of 69 against NIBRG-23 (A/turkey/Turkey/1/05; clade 2.2) and 42 against IBCDC-RG-2 (A/Indonesia/5/05-like; clade 2.1.3) while titres with aluminium hydroxide were <10. After the second immunization with CoVaccine HT™, GMT against NIBRG-23 was 599 and against IBCDC-RG-2 254, while those with aluminium hydroxide were 23 and 13, respectively. No local or systemic adverse events were detected in macaques. Safety of 5 µg H5N1 plus 0, 2 or 4 mg CoVaccine HT™ was investigated in a repeated dose study in rabbits. Groups of 6 or 9 male and female animals were immunized IM three times at three week intervals. None of the animals exerted treatment-related adverse reactions during the study or at necropsy 3 or 4 days after treatment. We concluded that a low dose of whole inactivated influenza virus plus CoVaccine HT™ is a promising, single-shot vaccine against pandemic influenza.

Evaluation of intravenous zanamivir against experimental influenza A (H5N1) virus infection in cynomolgus macaques.

We investigated the prophylactic and therapeutic efficacy of an intravenous (IV) formulation of zanamivir in a macaque infection model for highly pathogenic influenza A (H5N1) virus. Antiviral efficacy was dose-dependent, with no reduction in viral load observed at 2 mg/kg, but a significant reduction observed at 10 mg/kg (p=0.039) and at 20 mg/kg in the combined prophylactic and therapeutic groups (p=0.049) with both prophylaxis (commencing 12 h before infection) and therapy (commencing 4 h after infection) showing similar reductions in viral load. Combined gross pathology and microscopic pneumonia scores in the treated animals relative to untreated controls were significantly reduced at 10 mg/kg (p=0.02) and at 20 mg/kg in the prophylaxis group (p=0.02), but were not significant in the treatment group (p=0.145). In this new animal model for evaluation of influenza antivirals, despite variability observed between individual animals, IV zanamivir showed evidence of efficacy against highly pathogenic H5N1 virus.

In vitro processing and presentation of a lipidated cytotoxic T-cell epitope derived from measles virus fusion protein.

Lipopeptidic formulations have been described as efficient activators of cytotoxic T lymphocytes (CTL). To better understand the pathway via which lipopeptides reach the MHC class I molecules we studied the intracellular processing and presentation of a measles virus-derived CTL epitope, to which a palmitoyl moiety was added synthetically. The palmitoyl group was conjugated to the N-terminus either directly or via a spacer sequence. The use of single or double fluorescent-labeled lipopeptides allowed the visualization of intracellular processing of these antigens using confocal microscopy. Our data indicate that the spacer composition influences internalization of the conjugate into the cell, proteasomal degradation, translocation into the ER by the transporter associated with antigen processing (TAP), and the intracellular trafficking of lipopeptides.

Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques.

Modified vaccinia virus Ankara (MVA)-based recombinant viruses have been shown to be potent vaccine candidates for several infectious and neoplastic diseases. Since a major application of these live, replication-deficient vectors would be their use in immunocompromised or potentially immunocompromised individuals, a preclinical safety study was carried out. Macaques were inoculated with high doses of MVA (10(9)) via various routes, after immune-suppression by total-body irradiation, anti-thymocyte globulin treatment, or measles virus (MV) infection. No clinical, haematological or pathological abnormalities related to MVA inoculation were observed during a 13-day follow-up period. The presence of MVA genomes was demonstrated by nested PCR during the course of the experiment in all macaques, but from none of these animals replication competent MVA could be reisolated. These data suggest that MVA can safely be used as a basis for recombinant human vaccines, and that it is also safe for use in immunocompromised individuals.

Use of cotton rats for preclinical evaluation of measles vaccines.

The continued prevalence and medical impact of measles worldwide has created interest in the development of new generations of measles vaccines. Monkeys can be used for preclinical testing of these vaccines. However, a more practical and less expensive animal model is highly desirable, particularly for initial vaccine development and evaluation. Cotton rats have been shown to support the replication of different strains of measles virus (MV), and thus may be useful for these purposes. To test this concept, the immunogenicity and protective efficacy of two standard (Moraten and trivalent measles, mumps, rubella) and four experimental (two recombinant ALVAC, one ISCOM subunit and live attenuated Edmonston-Zagreb) MV vaccines were evaluated in naïve cotton rats, and cotton rats with passively acquired MV-specific neutralizing serum antibodies. All of the test vaccines were immunogenic and protected naíve animals from pulmonary infection and viral dissemination. However, under the conditions utilized, only the Edmonston-Zagreb vaccine provided such protection to animals with significant levels of passively acquired MV-specific neutralizing antibodies. The results of these tests and the potential of using cotton rats as an animal model for preliminary testing of MV vaccines are discussed.

In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components.

Semipurified Quil A and purified Quil A were used to prepare well-characterized subunit vaccine candidates against measles. Variation in the relative amounts of the measles virus (MV) fusion (F) protein, Quil A-components and lipids did not influence induction of antibody responses in mice, but had a pronounced effect on the capacity to induce cytotoxic T cell (CTL) activity of a CD8(+) MV F-protein specific human T cell clone in vitro. A characteristic MV iscom preparation based on the combined use of HPLC-purified Quil A-components QA-3 and QA-22 (QA-3/22) efficiently induced CTL activity in vitro. Comparable results were obtained by mixing beta-propiolactone inactivated MV with iscom-matrix QA-3/22 or free QA-22. On the basis of the data presented it was concluded that these three preparations are interesting MV vaccine candidates for further evaluation in pre-clinical experiments in a primate model.

Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies.

Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.

Solid-phase synthesis and application of double-fluorescent-labeled lipopeptides, containing a CTL-epitope from the measles fusion protein.

The mechanism which enables lipopeptides to induce cytotoxicity is not known. By preparing fluorescent-labeled lipopeptides one might unravel the mechanism of their entry into the cell and their intracellular pathway. A method of preparing double-fluorescent-labeled peptides by solid-phase chemistry is described. As model peptides we have chosen analogs of the sequence RRYPDAVYL, which occurs in the measles fusion protein (F438-446) and is an epitope for cytotoxic T lymphocytes. The peptides Pal-K(TMR)KKKRRYPDAVK(FL)L (7) and Pal-K(FL)KKKRRYPDAVK(TMR)L (8), in which Pal is palmitoyl and K(TMR) and K(FL) are Nepsilon-carboxytetramethylrhodamine- and Nepsilon-carboxyfluorescein-labeled lysyl residues, respectively, were prepared and obtained in approximately 30% yield after purification by high-performance liquid chromatography. The fluorescence of fluorescein and tetramethylrhodamine in lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L (7) was quenched to 98-99% due to intramolecular interaction of the labels. On incubation with trypsin (i.e. cleavage at the KKKRR-site) the fluorescence of both labels was restored. The intracellular routing of lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L was studied with human melanoma cell line, Mel/J, which was transfected with human leukocyte antigen B*2705. It appeared that the double-fluorescent-labeled lipopeptide was able to induce antigen-specific cytotoxicity. Furthermore, preliminary confocal microscopical studies indicated that this lipopeptide is observed intracellularly.