Stittelaar, K.J.

Consecutive CT in vivo lung imaging as quantitative parameter of influenza vaccine efficacy in the ferret model (Article)

2012-12-07T00:00:00Z

Preclinical vaccine efficacy studies are generally limited to certain read out parameters such as assessment of virus titers in swabs and organs, clinical signs, serum antibody titers, and pathological changes. These parameters are not always routinely applied and not always scheduled in a logical standardized way. We used computed tomography (CT) imaging as additional and novel read out parameter in a vaccine efficacy study by quantifying alterations in aerated lung volumes in ferrets challenged with the 2009 pandemic A/H1N1 influenza virus. Vaccination protected from marked variations in aerated lung volumes compared to naive controls. The vaccinated group showed a daily gradual mean reduction with a maximum of 7.8%, whereas the controls showed a maximum of 14.3% reduction. The pulmonary opacities evident on CT images were most pronounced in the placebo-treated controls, and corresponded to significantly increased relative lung weights at necropsy. This study shows that consecutive in vivo CT imaging allows for a day to day read out of vaccine efficacy by quantification of altered aerated lung volumes.

Comparison of temporal and spatial dynamics of seasonal H3N2, pandemic H1N1 and highly pathogenic avian influenza H5N1 virus infections in ferrets (Article)

2012-08-08T00:00:00Z

Humans may be infected by different influenza A viruses-seasonal, pandemic, and zoonotic-which differ in presentation from mild upper respiratory tract disease to severe and sometimes fatal pneumonia with extra-respiratory spread. Differences in spatial and temporal dynamics of these infections are poorly understood. Therefore, we inoculated ferrets with seasonal H3N2, pandemic H1N1 (pH1N1), and highly pathogenic avian H5N1 influenza virus and performed detailed virological and pathological analyses at time points from 0.5 to 14 days post inoculation (dpi), as well as describing clinical signs and hematological parameters. H3N2 infection was restricted to the nose and peaked at 1 dpi. pH1N1 infection also peaked at 1 dpi, but occurred at similar levels throughout the respiratory tract. H5N1 infection occurred predominantly in the alveoli, where it peaked for a longer period, from 1 to 3 dpi. The associated lesions followed the same spatial distribution as virus infection, but their severity peaked between 1 and 6 days later. Neutrophil and monocyte counts in peripheral blood correlated with inflammatory cell influx in the alveoli. Of the different parameters used to measure lower respiratory tract disease, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups. There was extra-respiratory spread to more tissues-including the central nervous system-for H5N1 infection than for pH1N1 infection, and to none for H3N2 infection. This study shows that seasonal, pandemic, and zoonotic influenza viruses differ strongly in the spatial and temporal dynamics of infection in the respiratory tract and extra-respiratory tissues of ferrets.

Modification of the Ferret Model for Pneumonia From Seasonal Human Influenza A Virus Infection (Article)

2012-05-01T00:00:00Z

The primary complication of seasonal influenza in humans is viral pneumonia. A conventional animal model-intranasal inoculation of ferrets with 106median 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 109and 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.

Efficacy of live attenuated vaccines against 2009 pandemic H1N1 influenza in ferrets (Article)

2011-11-15T00:00:00Z

The advent of the H1N1 influenza pandemic (pH1N1) in 2009 triggered the rapid production of pandemic influenza vaccines, since seasonal influenza vaccines were expected and demonstrated not to provide significant cross-protection against the newly emerged pandemic virus. To increase vaccine production capacity and further evaluate the effectiveness of different candidate pandemic influenza vaccines, the World Health Organization stimulated the evaluation of different vaccination concepts including the use of live attenuated influenza vaccines (LAIVs). Therefore, we have immunized ferrets intranasally with a single dose of pH1N1-LAIV from different manufacturers. They all induced adequate serum HI antibody titers in the ferrets and protected them against intratracheal wild-type pH1N1 virus challenge: pH1N1 virus replication in the upper respiratory tract and lungs was reduced and no disease signs or severe broncho-interstitial pneumonia were observed in any of the vaccinated ferrets. These data together with the relatively efficient production process emphasize the potential of the LAIV concept for pandemic preparedness.

Multidrug resistant 2009 a/h1n1 influenza clinical isolate with a neuraminidase i223r mutation retains its virulence and transmissibility in ferrets (Article)

2011-09-01T00:00:00Z

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

Pulmonary pathology of pandemic influenza A/H1N1 virus (2009)-infected ferrets upon longitudinal evaluation by computed tomography (Article)

2011-08-01T00:00:00Z

We investigated the development of pulmonary lesions in ferrets by means of computed tomography (CT) following infection with the 2009 pandemic A/H1N1 influenza virus and compared the scans with gross pathology, histopathology and immunohistochemistry. Groundglass opacities observed by CT scanning in all infected lungs corresponded to areas of alveolar oedema at necropsy. These areas were most pronounced on day 3 and gradually decreased from days 4 to 7 post-infection. This pilot study shows that the non-invasive imaging procedure allows quantification and characterization of influenza-induced pulmonary lesions in living animals under biosafety level 3 conditions and can thus be used in pre-clinical pharmaceutical efficacy studies.

Efficacy of vaccination with different combinations of MF59-adjuvanted and nonadjuvanted seasonal and pandemic influenza vaccines against pandemic H1N1 (2009) influenza virus infection in ferrets (Article)

2011-03-01T00:00:00Z

Serum antibodies induced by seasonal influenza or seasonal influenza vaccination exhibit limited or no cross-reactivity against the 2009 pandemic swine-origin influenza virus of the H1N1 subtype (pH1N1). Ferrets immunized once or twice with MF59-adjuvanted seasonal influenza vaccine exhibited significantly reduced lung virus titers but no substantial clinical protection against pH1N1-associated disease. However, priming with MF59-adjuvanted seasonal influenza vaccine significantly increased the efficacy of a pandemic MF59-adjuvanted influenza vaccine against pH1N1 challenge. Elucidating the mechanism involved in this priming principle will contribute to our understanding of vaccine- and infection-induced correlates of protection. Furthermore, a practical consequence of these findings is that during an emerging pandemic, the implementation of a priming strategy with an available adjuvanted seasonal vaccine to precede the eventual pandemic vaccination campaign may be useful and life-saving. Copyright

Pandemic H1N1 vaccine requires the use of an adjuvant to protect against challenge in naïve ferrets (Article)

2011-02-02T00:00:00Z

In the context of an A/H1N1 influenza pandemic situation, this study demonstrates that heterologous vaccination with an AS03-adjuvanted 2008/2009 seasonal trivalent and pandemic H5N1 monovalent split vaccine conferred partial protection in influenza-naïve ferrets after challenge with the influenza pandemic H1N1 A/The Netherlands/602/09 virus. Further, unlike saline control and non-adjuvanted vaccine, it was shown that immunization of naïve ferrets with an AS03-adjuvanted pandemic H1N1 A/California/7/09 influenza split vaccine induced increased antibody response and enhanced protection against the challenge strain, including significant reduction in viral shedding in the upper respiratory tract and reduced lung pathology post-challenge. These results show the need for vaccination with the adjuvanted vaccine to fully protect against viral replication and influenza disease in unprimed ferrets.

Longevity of the protective immune response induced after vaccination with one or two doses of AS03A-adjuvanted split H5N1 vaccine in ferrets (Article)

2011-02-01T00:00:00Z

It is crucial that a safe and effective pandemic vaccine be rapidly available to combat a new pandemic threat. In this study we investigated the magnitude and persistence of the protective efficacy induced by one or two doses (3.75μg HA/dose) of AS03A-adjuvanted H5N1 A/Indonesia/5/05 split vaccine in a lethal ferret challenge model. All ferrets that received at least one dose of adjuvanted vaccine 4 weeks before homologous challenge survived and showed reduced or undetectable virus replication in the lungs and the upper airways. Ferrets receiving two doses of adjuvanted vaccine 19 and 16 weeks before the challenge also showed high level of protection from replication in the lungs and the upper airways, albeit with only 83% survival. Animals in the control groups (non-adjuvanted vaccine or saline) and animals immunized with one dose of adjuvanted vaccine administered 10 or 16 weeks before challenge showed only 17-33% survival rate after challenge. In conclusion, our observations support the possibility that a single dose of AS03A-adjuvanted H5N1 split vaccine can offer a rapid and short term but partial protection against disease. A second dose of the adjuvanted vaccine, which can be given with a flexible injection schedule, was shown to be essential to induce appreciable levels of antibodies and long-term protection.

Feasibility of single-shot H5N1 influenza vaccine in ferrets, macaques and rabbits (Article)

2010-11-29T00:00:00Z

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.

Experimental pandemic (H1N1) 2009 virus infection of cats (Article)

2010-10-01T00:00:00Z

To demonstrate that pandemic (H1N1) 2009 virus may cause respiratory disease in cats, we intratracheally infected cats. Diffuse alveolar damage developed. Seroconversion of sentinel cats indicated cat-to-cat virus transmission. Unlike in cats infected with highly pathogenic avian influenza virus (H5N1), extrarespiratory lesions did not develop in cats infected with pandemic (H1N1) 2009 virus.

Cross-clade immunity in cats vaccinated with a canarypox-vectored avian influenza vaccine (Article)

2010-07-01T00:00:00Z

Several felid species have been shown to be susceptible to infection with highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype. Infection of felids by H5N1 HPAI virus is often fatal, and cat-to-cat transmission has been documented. Domestic cats may then be involved in the transmission of infection to other animals but also to humans. A particular concern is the hypothetical role of the cat in the adaptation of the virus to mammalian species, thus increasing the pandemic risk. Therefore, the development of a HPAI vaccine for domestic cats should be considered a veterinary and also a public health priority. Here we show that vaccination of cats with a recombinant canarypox (ALVAC®1) virus, expressing the hemagglutinin (HA) of influenza virus A/chicken/Indonesia/03 (H5N1) confers protection against challenge infection with two antigenically distinct H5N1 virus isolates from humans. Despite low hemagglutination inhibiting (HI) antibody titers at the time of challenge, all vaccinated cats were protected against mortality and had reduced histopathological changes in the lungs. Importantly, viral shedding was reduced in vaccinated cats as compared to controls, suggesting that vaccination of cats could reduce the risk of viral transmission. In conclusion this study showed that the recombinant canarypox virus protected cats against homologous and heterologous H5N1 HPAI virus challenges.

Severity of pneumonia due to new H1N1 influenza virus in ferrets is intermediate between that due to seasonal H1N1 virus and highly pathogenic avian influenza H5N1 virus (Article)

2010-04-01T00:00:00Z

Background. The newly emerged influenza A(H1N1) virus (new H1N1 virus) is causing the first influenza pandemic of this century. Three influenza pandemics of the previous century caused variable mortality, which largely depended on the development of severe pneumonia. However, the ability of the new H1N1 virus to cause pneumonia is poorly understood. Methods. The new H1N1 virus was inoculated intratracheally into ferrets. Its ability to cause pneumonia was compared with that of seasonal influenza H1N1 virus and highly pathogenic avian influenza (HPAI) H5N1 virus by using clinical, virological, and pathological analyses. Results. Our results showed that the new H1N1 virus causes pneumonia in ferrets intermediate in severity between that caused by seasonal H1N1 virus and by HPAI H5N1 virus. The new H1N1 virus replicated well throughout the lower respiratory tract and more extensively than did both seasonal H1N1 virus (which replicated mainly in the bronchi) and HPAI H5N1 virus (which replicated mainly in the alveoli). High loads of new H1N1 virus in lung tissue were associated with diffuse alveolar damage and mortality. Conclusions. The new H1N1 virus may be intrinsically more pathogenic for humans than is seasonal H1N1 virus.

New class of monoclonal antibodies against severe influenza: Prophylactic and therapeutic efficacy in ferrets (Article)

2010-02-08T00:00:00Z

Background: The urgent medical need for innovative approaches to control influenza is emphasized by the widespread resistance of circulating subtype H1N1 viruses to the leading antiviral drug oseltamivir, the pandemic threat posed by the occurrences of human infections with highly pathogenic avian H5N1 viruses, and indeed the evolving swine-origin H1N1 influenza pandemic. A recently discovered class of human monoclonal antibodies with the ability to neutralize a broad spectrum of influenza viruses (including H1, H2, H5, H6 and H9 subtypes) has the potential to prevent and treat influenza in humans. Here we report the latest efficacy data for a representative antibody of this novel class. Methodology/Principal Findings: We evaluated the prophylactic and therapeutic efficacy of the human monoclonal antibody CR6261 against lethal challenge with the highly pathogenic avian H5N1 virus in ferrets, the optimal model of human influenza infection. Survival rates, clinically relevant disease signs such as changes in body weight and temperature, virus replication in lungs and upper respiratory tract, as well as macro- and microscopic pathology were investigated. Prophylactic administration of 30 and 10 mg/kg CR6261 prior to viral challenge completely prevented mortality, weight loss and reduced the amount of infectious virus in the lungs by more than 99.9%, abolished shedding of virus in pharyngeal secretions and largely prevented H5N1-induced lung pathology. When administered therapeutically 1 day after challenge, 30 mg/kg CR6261 prevented death in all animals and blunted disease, as evidenced by decreased weight loss and temperature rise, reduced lung viral loads and shedding, and less lung damage. Conclusions/Significance: These data demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant animal model of influenza and justify clinical development of this approach as intervention for both seasonal and pandemic influenza.

RNA secondary structures in the proximal 3′UTR of Indonesian Dengue 1 virus strains (Article)

2009-06-01T00:00:00Z

The characteristics of DENV-1 viruses, isolated during the 2001-2002 outbreak in Indonesia were studied. The secondary structure of the 3′UTR of different DENV-1 strains derived from Indonesian patients was compared with the 3′UTR of previously described DENV-1 sequences. The complete 3′UTR of DENV-1 was sequenced from 13 patients suffering from the severe form of dengue virus infection (dengue hemorrhagic fever). Prediction of RNA secondary structure of the 3′UTR revealed some previously unidentified conserved structures in the proximal region of the 3′UTR, the role of which in viral replication is still unknown. In addition our data suggest that some structural elements previously described in the distal part of the 3′UTR are partly dependent on the proximal part of the UTR. Our data support the existence of previously unidentified conserved secondary structures in the proximal part of the 3′UTR and their roles need to be further investigated.

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

2008-11-01T00:00:00Z

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.

Measles vaccination: New strategies and formulations (Article)

2008-10-01T00:00:00Z

Measles is a highly contagious viral disease. With 1 million deaths reported in 1996, measles was the leading cause of vaccine-preventable deaths. However, in recent years, significant progress has been made in measles control, reducing deaths attributed to measles to 454,000 in 2004 and 242,000 in 2006. The main strategy behind this reduction has been the improvement of vaccination coverage and implementation of a second opportunity for immunization with the live-attenuated measles vaccine. The Measles Initiative, a partnership between the American Red Cross, CDC, UNICEF, WHO and UN Foundation, has had a significant role in this achievement. Here, we provide an overview of old and new vaccination strategies, and discuss changes in the route of administration of the existing live-attenuated vaccine, the development of new-generation nonreplicating measles virus vaccine candidates and attempts to use recombinant measles virus as a vector for vaccination against other pathogens.

Cross-protection against lethal H5N1 challenge ferrets with an adjuvanted pandemic influenza vaccine (Article)

2008-01-02T00:00:00Z

Background. Unprecedented spread between birds and mammals of highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtype has resulted in hundreds of human infections with a high fatality rate. This has highlighted the urgent need for the development of H5N1 vaccines that can be produced rapidly and in sufficient quantities. Potential pandemic inactivated vaccines will ideally induce substantial intra-subtypic cross-protection in humans to warrant the option of use, either prior to of just after the start of a pandemic outbreak. In the present study, we evaluated a split H5N1 A/H5N1/Vietnam/1194/04, clade 1 candidate vaccine, adjuvanted with a proprietary oil-in- water emulsion based Adjuvant System proven to be well-tolerated and highly immunogenic in the human (Leroux-Roels et al. (2007) The Lancet 370:580-589), for its ability to induce intra-subtypic cross-protection against clade 2 H5N1/A/Indonesia/5/05 challenge in ferrets. Methodology and Principal Findings. All ferrets in control groups receiving non-adjuvanted vaccine or adjuvant alone failed to develop specific or cross-reactive neutralizing antibodies and all died or had to be euthanized within four days of virus challenge. Two deses of adjuvanted split H5N1 vaccine containing ≥ 1.7 μg HA induced neutralizing antibodies in the majority of ferrets to both clade 1 (17/ 23 (74%) responders) and clade 2 viruses (14/23 (61%) responders), and 96% (22/23) of vaccinees survived the lethal challenge. Furthermore lung virus loads and viral shedding in the upper respiratory tract were reduced in vaccinated animals relative to controls suggesting that vaccination might also confer a reduced risk of viral transmission. Conclusion. These protection data in a stringent challenge model in association with an excellent clinical profile highlight the potential of this adjuvanted H5N1 candidate vaccine as an effective tool in pandemic preparedness.

Efficacy of a live attenuated tetravalent candidate dengue vaccine in naïve and previously infected cynomolgus macaques (Article)

2007-07-20T00:00:00Z

The development of a safe and effective vaccine against dengue is a public health priority. Attempts to evaluate candidate vaccine formulations in human volunteers were largely unsuccessful, at least in part due to too high reactogenicity of some of the candidate vaccines tested. We evaluated a live attenuated tetravalent dengue vaccine candidate in flavivirus naïve and dengue virus type 3 immune non-human primates. Immune responses were measured both at the humoral and the cellular level and the efficacy of this vaccine candidate was evaluated by challenging the vaccinated animals with dengue virus type 4. Humoral and cellular immune responses upon vaccination were similar to those described after natural infection in humans. All animals were protected from developing viremia upon challenge infection. In addition, primary dengue virus type 3 infection of macaques neither influenced the immune response upon vaccination, nor interfered with vaccine-induced protection from dengue virus type 4 challenge infection. The data suggest that the live attenuated tetravalent vaccine candidate used is promising and warrant further safety and efficacy testing in clinical trials.

Characterization of humoral and cellular immune responses in cynomolgus macaques upon primary and subsequent heterologous infections with dengue viruses (Article)

2007-07-01T00:00:00Z

Since studying the pathogenesis of dengue virus associated disease in humans has several limitations, an appropriate animal model is needed. Therefore, we investigated kinetics of viremia as well as humoral and cellular immune responses, after primary, secondary and tertiary heterologous dengue virus infections in cynomolgus macaques: these parameters were largely similar to those observed in natural human infection upon primary infection. Both antibody and T-cell responses measured were largely cross-reactive. Upon secondary infection with a heterologous virus serotype, T-cell responses specific for the primary infecting serotype were more pronounced, especially when the immune system was primed with dengue 1 virus. Measurement of transcription levels of pro- and anti-inflammatory cytokines in white blood cells upon primary and secondary infection generally showed a balanced response. In addition, a region of the NS2A protein of dengue viruses was identified that induces T-cell responses in macaques.

Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus. (Article)

2005-06-01T00:00:00Z

The use of classical smallpox vaccines based on vaccinia virus (VV) is associated with severe complications in both naive and immune individuals. Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of VV, has been proven to be safe in humans and immunocompromised animals, and its efficacy against smallpox is currently being addressed. Here we directly compare the efficacies of MVA alone and in combination with classical VV-based vaccines in a cynomolgus macaque monkeypox model. The MVA-based smallpox vaccine protected macaques against a lethal respiratory challenge with monkeypox virus and is therefore an important candidate for the protection of humans against smallpox.

Longevity of neutralizing antibody levels in macaques vaccinated with Quil A-adjuvanted measles vaccine candidates (Article)

2002-12-13T00:00:00Z

Quil A-based candidate measles vaccines have been shown to be immunogenic and protective in cotton rats and macaques. Here we studied the longevity of protective VN antibody levels induced in macaques with one dose of measles virus (MV) iscom. Inactivated MV adjuvanted with iscom-matrix or with purified Quillaja saponin QA-22 were also tested. All animals developed high levels of VN antibody and MV-specific IFNγ-producing cells. Especially the high VN antibody levels induced by the latter two preparations showed virtually no decrease during the 2-year follow-up. These highly promising candidate MV vaccines should now be tested in infant macaques in the presence or absence of passively transferred and/or maternally derived VN antibodies. In addition, the immunopathological safety of the constructs should be evaluated in the atypical measles model in rhesus macaques.

Vaccination against measles: a neverending story. (Article)

2002-08-01T00:00:00Z

Measles, a highly contagious viral disease, is a major childhood killer in developing countries, accounting for almost 1 million deaths every year globally. Measles virus normally does not cause a persistent infection, no animal reservoir for measles virus exists, no vector is involved in its spread, only one serotype exists, the virus is antigenically stable and vaccination with the currently used live attenuated vaccines proved to be highly effective in preventing disease. Therefore, theoretically measles should be considered eradicable. This article provides a review of past and current measles vaccination efforts and development and need of new generation experimental measles vaccines.

Comparison of the efficacy of early versus late viral proteins in vaccination against SIV. (Article)

2002-07-26T00:00:00Z

The immune response against early regulatory proteins of simian- and human immunodeficiency virus (SIV, HIV) has been associated with a milder course of infection. Here, we directly compared vaccination with Tat/Rev versus Pol/Gag. Challenge infection with SIVmac32H (pJ5) suggested that vaccination with Tat/Rev induced cellular immune responses that enabled cynomolgus macaques to more efficiently control SIV replication than the vaccine-induced immune responses against Pol/Gag. Vaccination with Tat/Rev resulted in reduced plasma SIV loads compared with control (P=0.058) or Pol/Gag-vaccinated (P=0.089) animals, with undetectable plasma viral loads in two of the four Tat/Rev-vaccinated animals. Therefore, the results warrant further investigation of the early regulatory proteins and their potential for vaccination against HIV.

Enteric administration of a live attenuated measles vaccine does not induce protective immunity in a macaque model (Article)

2002-07-26T00:00:00Z

To test the option of oral vaccination with a live attenuated measles vaccine (LAV), we have evaluated the potential of an orally administered enteric-coated tablet containing a candidate LAV (strain Leningrad-16, MV-L16). To this end three groups of two cynomolgus macaques each were vaccinated via different routes with 103.8TCID50MV-L16 vaccine: intramuscularly (i.m.), intraintestinally (i.i.) upon laparotomy and via enteric-coated tablets. Upon vaccination, MV-L16 could only be isolated from one of the i.m.-vaccinated monkeys and not from any of the other five. Both the i.m.-infected monkeys and one of the i.i.-infected monkeys developed a MV-specific serum antibody response. Also, MV-specific CD8+IFNγ-producing T cells could be demonstrated in all three monkeys that had seroconverted. Upon challenge with wild-type MV 1 year after vaccination, only these three monkeys proved to be protected. These data do not support the viability of the concept of oral vaccination with LAVs.

Priming of measles virus-specific humoral- and cellular-immune responses in macaques by DNA vaccination (Article)

2002-05-15T00:00:00Z

Although the currently used live attenuated measles vaccines are safe and effective, they are dependent on cold chain maintenance and are often ineffective in young infants due to interference by maternal antibody. Therefore, besides vector-based vaccines, different new generation non-replicating candidate measles vaccines are being considered, including nucleic acid vaccines. We have vaccinated cynomolgus macaques transdermally with DNA plasmids encoding measles virus (MV) proteins. Following two vaccinations, low serum antibody responses were detected. Wild-type measles virus challenge 1 year after vaccination showed reduced viraemia in some animals. However, accelerated humoral- and cellular-immune responses were observed in all vaccinated macaques, demonstrating successful priming by the DNA vaccines.

Vaccination against Measles: evaluation of novel approaches (Doctoral Thesis)

2001-12-20T00:00:00Z

Measles, also called morbilli or rubeola, is a highly contagious disease of humans. After an incubation period of 9-11 days characteristic clinical signs develop like coryza, cervical lymphadenitis, so-called Koplik's spots in the mouth, conjunctivitis, photophobia, myalgia, malaise, sneezing and coughing lasting for about 1 week. Fever precedes the typical rash consisting of generalised maculopapular lesions that first appear on the face and soon after on the trunk and the extremities. Measles is one out of six infectious diseases that cause 90% of infectious disease deaths, worldwide (Fig.la). It is a major childhood killer in developing countries, accounting for about 900,000 deaths a year (Fig.1 b). Data from the pre-vaccination era showed that in Europe by 10 years of age virtually everyone had evidence of past measles virus (MV) infection.

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

2001-10-12T00:00:00Z

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.

MVA: a cuckoo in the vaccine nest? [Editorial] (Article)

2001-06-14T00:00:00Z

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

2001-06-14T00:00:00Z

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 (109) 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. (Article)

2000-08-15T00:00:00Z

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 naïve 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.

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

2000-05-02T00:00:00Z

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.

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

2000-05-01T00:00:00Z

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 (Article)

2000-01-01T00:00:00Z