Comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys

Three antigenic chimeric live attenuated tick-borne encephalitis virus (TBEV) vaccine candidates were compared for level of replication in murine and human neuroblastoma cells, for neurovirulence and neuroinvasiveness in mice, and for safety, immunogenicity and efficacy in rhesus monkeys. Two chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue type 4 virus (DEN4) with the corresponding genes of a Far Eastern TBEV, Sofjin strain, in the presence (TBEV/DEN4Delta30) or absence (TBEV/DEN4) of a 30 nucleotide deletion (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome. A third chimeric TBEV vaccine candidate was based on the antigenically distant, but naturally attenuated Langat virus (LGT). Chimerization of LGT with DEN4 resulted in decreased neurovirulence and neuroinvasiveness in mice and highly restricted viremia in rhesus monkeys. Also, the LGT/DEN4 chimera was highly restricted in replication in both murine and human neuroblastoma cells. In contrast, TBEV/DEN4 and TBEV/DEN4Delta30 were neither attenuated for neurovirulence in the mice nor restricted in replication in the neuroblastoma cells. However, both were highly attenuated for neuroinvasiveness in mice. TBEV/DEN4 replicated to moderately high titer in rhesus monkeys (mean peak viremia=10(3.1)PFU/ml) indicating that the TBEV/DEN4 chimerization had only a modest, if any, attenuating effect in monkeys. However, the addition of the Delta30 mutation to TBEV/DEN4 greatly attenuated the chimeric virus for rhesus monkeys (mean peak viremia=10(0.7)PFU/ml) and induced a higher level of antibody against the TBEV than did LGT/DEN4. A single dose of either highly attenuated TBEV/DEN4Delta30 or LGT/DEN4 vaccine candidate or three doses of an inactivated TBEV vaccine were efficacious in monkeys against wild-type LGT challenge. These results indicate that both TBEV/DEN4Delta30 and LGT/DEN4 are safe and efficacious in rhesus monkeys and should be further evaluated as vaccine candidates for use in humans.     

Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge

Dengue epidemics increasingly pose a public health problem in most countries of the tropical and subtropical areas. Despite decades of research, development of a safe and effective live dengue virus vaccine is still at the experimental stage. To explore an alternative vaccine strategy, we employed the highly attenuated, replication-deficient modified vaccinia Ankara (MVA) as a vector to construct recombinants for expression of the major envelope glycoprotein of one or more dengue virus serotypes. MVA recombinants expressing the highly immunogenic C-terminally truncated dengue type 2 virus (DEN2) or dengue type 4 virus (DEN4) envelope protein (E), approx. 80% of the full-length, were evaluated for their protective immunity in animal models. Each of these recombinants elicited an elevated antibody response to DEN2 or DEN4 E in mice following the booster inoculation, as detected by radio-immunoprecipitation. Recombinant MVA-DEN2 80%E, but not MVA-DEN4 80%E, induced a neutralizing antibody response. The MVA-DEN2 80%E recombinant was chosen to further evaluate its ability to induce resistance to wild type DEN2 challenge in monkeys. Monkeys immunized twice with recombinant MVA-DEN2 80%E developed a low to moderate antibody response and were partially protected against DEN2 challenge, as determined by the viremia pattern. Importantly, the subsequent study showed that all four monkeys immunized with the recombinant in a three dose schedule developed an increased level of antibodies and were completely protected against DEN2 challenge. The potential efficacy of recombinant MVA-DEN2 80%E to protect primates against dengue infection suggests that construction and evaluation of MVA recombinants expressing other serotypes of dengue virus E for use in a tetravalent vaccine strategy might be warranted.     

Vaccination of volunteers with low-dose,live-attenuated,dengue viruses leads to serotype-specific immunologic and virologic profiles

There are currently no vaccines or therapeutics to prevent dengue disease which ranges in severity from asymptomatic infections to life-threatening illness. The National Institute of Allergy and Infectious Diseases (NIAID) Division of Intramural Research has developed live, attenuated vaccines to each of the four dengue serotypes (DENV-1–DENV-4). Two doses (10 PFU and 1000 PFU) of three monovalent vaccines were tested in human clinical trials to compare safety and immunogenicity profiles. DEN4Δ30 had been tested previously at multiple doses. The three dengue vaccine candidates tested (DEN1Δ30, DEN2/4Δ30, and DEN3Δ30/31) were very infectious, each with a human infectious dose 50% ≤ 10 PFU. Further, infectivity rates ranged from 90 to 100% regardless of dose, excepting DEN2/4Δ30 which dropped from 100% at the 1000 PFU dose to 60% at the 10 PFU dose. Mean geometric peak antibody titers did not differ significantly between doses for DEN1Δ30 (92 ± 19 vs. 214 ± 97, p = 0.08); however, significant differences were observed between the 10 PFU and 1000 PFU doses for DEN2/4Δ30, 19 ± 9 vs. 102 ± 25 (p = 0.001), and DEN3Δ30/31, 119 ± 135 vs. 50 ± 50 (p = 0.046). No differences in the incidences of rash, neutropenia, or viremia were observed between doses for any vaccines, though the mean peak titer of viremia for DEN1Δ30 was higher at the 1000 PFU dose (0.5 ± 0 vs. 1.1 ± 0.1, p = 0.007). These data demonstrate that a target dose of 1000 PFU for inclusion of each dengue serotype into a tetravalent vaccine is likely to be safe and generate a balanced immune response for all serotypes.     

The live attenuated chimeric vaccine rWN/DEN4Δ30 is well-tolerated and immunogenic in healthy flavivirus-naïve adult volunteers

WNV has become the leading vector-borne cause of meningoencephalitis in the United States. Although the majority of WNV infections result in asymptomatic illness, approximately 20% of infections result in West Nile fever and 1% in West Nile neuroinvasive disease (WNND), which causes encephalitis, meningitis, or flaccid paralysis. The elderly are at particular risk for WNND, with more than half the cases occurring in persons older than sixty years of age. There is no licensed treatment for WNND, nor is there any licensed vaccine for humans for the prevention of WNV infection. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a recombinant live attenuated WNV vaccine based on chimerization of the wild-type WNV NY99 genome with that of the live attenuated DENV-4 candidate vaccine rDEN4Δ30. The genes encoding the prM and envelope proteins of DENV-4 were replaced with those of WNV NY99 and the resultant virus was designated rWN/DEN4Δ30. The vaccine was evaluated in healthy flavivirus-naïve adult volunteers age 18–50 years in two separate studies, both of which are reported here. The first study evaluated 10³ or 10⁴ PFU of the vaccine given as a single dose; the second study evaluated 10⁵ PFU of the vaccine given as two doses 6 months apart. The vaccine was well-tolerated and immunogenic at all three doses, inducing seroconversion to WNV NY99 in 74% (10³ PFU), 75% (10⁴ PFU), and 55% (10⁵ PFU) of subjects after a single dose. A second 10⁵ PFU dose of rWN/DEN4Δ30 given 6 months after the first dose increased the seroconversion rate 89%. Based on the encouraging results from these studies, further evaluation of the candidate vaccine in adults older than 50 years of age is planned.     

Chimeric West Nile/dengue virus vaccine candidate: preclinical evaluation in mice, geese and monkeys for safety and immunogenicity

A live attenuated virus vaccine is being developed to protect against West Nile virus (WN) disease in humans. Previously, it was found that chimeric West Nile/dengue viruses (WN/DEN4 and WN/DEN4Delta30) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue type 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome were attenuated and efficacious in mice and monkeys against WN challenge. Here, we report the generation of a clinical lot of WN/DEN4Delta30 virus and its further preclinical evaluation for safety and immunogenicity in mice, geese and monkeys. The vaccine candidate had lost neuroinvasiveness in highly sensitive immunodeficient mice inoculated intraperitoneally and had greatly reduced neurovirulence in suckling mice inoculated intracerebrally (IC). Compared to the wild-type WN parent, the chimeric virus was highly restricted in replication in both murine and human neuroblastoma cells as well as in brains of suckling mice. The WN/DEN4Delta30 virus failed to infect geese, indicating that chimerization of WN with DEN4 completely attenuated WN for this avian host. This observation suggests that the WN/DEN4 chimeric viruses would be restricted in their ability to be transmitted from vaccinees to domestic or wild birds. In monkeys, the WN/DEN4Delta30 vaccine candidate was highly immunogenic despite its low level of replication with undetectable viremia. Furthermore, the WN/DEN4Delta30 vaccine virus was safe and readily induced neutralizing antibodies against WN in monkeys immune to each of the four serotypes of dengue virus. These studies confirm the attenuation of WN/DEN4Delta30 for non-human primates, including dengue-immune monkeys, and demonstrate both a highly restricted replication (>10(8)-fold decrease) in the brain of mice inoculated IC and an absence of infectivity for birds, findings that indicate this vaccine should be safe for both the recipient and the environment.     

云南省西南边境地区人血清虫媒病毒抗体调查

本文报告了云南省西南部4个专州9个县的760份人血清对11种虫媒病毒抗原的血凝抑制抗体检查结果,发现不仅乙脑和登革抗体较普遍,同时也存在其它黄病毒组和甲组虫媒病毒的抗体。
甲组虫媒病毒抗体阳性率平均为36.18%(275/760),其中MAY阳性率最高占总阳性数的68.73%(189/275),其次是CHIK占22.18%(61/275),几何平均滴度(GMT),VEE为164.4,MAY为94.48,CHIK为66.7。有交叉反应的血清178份,占64.73%.
黄病毒组抗体阳性率为77.37%(588/760),其中登革和乙脑阳性率最高,前者为36.58%(278/760,后者为27.89%(212/760),其它病毒的阳性率分别为:KFD22.24%(169/760),MVE22.11%(168/760),KUN18.68%(142/760),POW8.82%(67/760),LGT7.89%(60/760)。黄病毒抗体检查中重复感染者较多,占检查数的33.03%(251/760),占黄病毒组总阳性数的42.69%(251/588)。抗体滴度在1:640以上者有403人,占总阳性人次数的36.64%(403/1100),几何平均滴度为355.8。组内交叉反应率达97.09%。     

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.     

Induction of protective immunity against Dengue virus type 2: comparison of candidate live attenuated and recombinant vaccines

Dengue (DEN) viruses (serotypes 1 to 4) are mosquito-borne flaviviruses which cause about fifty million human infections annually and represent an expanding public health problem in the tropics. At present, there are no safe and effective vaccines which induce protective immunity to all four serotypes of DEN. Natural infection or vaccination with native and recombinant proteins may induce an immune response to the surface envelope E-protein which was shown to be protective to super-infection with homologous serotype of the virus. Purified recombinant E-protein was made in the baculovirus-Spodoptera frugiperda expression system. This protein induced neutralizing antibodies in mice. These results prompted us to immunize cynomolgus monkeys (Macaca fascicularis) with either a live attenuated DEN-2 vaccine or the recombinant E-protein complexed to aluminum hydroxide. After immunization, the monkeys were challenged with the homologous DEN virus. Serum was collected at several time points and a virus-specific antibody response including a virus neutralizing antibody response was measured. Antibody kinetics and levels were similar to those recorded in humans with a natural DEN-virus infection. Virus isolation and type specific RT-PCR were performed on the serum samples. The virus was isolated from sham vaccinated control monkeys but not from monkeys vaccinated with the live attenuated vaccine. One of the two monkeys immunized with the recombinant E-protein was also protected. Taken together these data indicate the potential of both candidate vaccines and stress the need for evaluation of different antigen presentation systems for the development of a subunit vaccine approach for DEN.     

Development and testing of a new tick-borne encephalitis virus vaccine candidate for veterinary use

In tick-borne encephalitis (TBE) endemic areas, consumption of unpasteurized milk or milk products from grazing domestic ruminants (goats, cattle, and sheep) represents a risk of TBE virus (TBEV) infection for humans. In addition to vaccination of humans, human alimentary TBEV infections can be avoided by pasteurizing milk or by vaccination of the ruminants. However, there is presently no TBEV vaccine for veterinary use. Here, we developed a new veterinary TBE vaccine candidate based on cell culture-derived, purified, and formaldehyde-inactivated TBEV (strain Hypr). The safety and immunogenicity of the vaccine was evaluated in mice and sheep and was well-tolerated while eliciting the production of high levels of virus-neutralizing antibodies. Vaccination provided full protection against lethal TBE in mice, prevented development of viremia in sheep and presence of TBEV in milk of lactating ewes. This vaccine is a good candidate for immunization of ruminants to prevent alimentary milk-borne TBEV infections in humans.     

Clinical proof of principle for ChimeriVax: recombinant live, attenuated vaccines against flavivirus infections.

ChimeriVax is a live, attenuated recombinant virus constructed from yellow fever (YF) 17D in which the envelope protein genes of YF 17D are replaced with the corresponding genes of another flavivirus. A ChimeriVax vaccine was developed against Japanese encephalitis (JE). A randomized, double-blind, outpatient study was conducted to compare the safety and immunogenicity of ChimeriVax-JE and YF 17D. Six YF immune and six non-immune adults were randomized to receive a single SC inoculation of ChimeriVax-JE (5log(10)PFU), ChimeriVax-JE (4log(10)PFU) or YF-VAX((R)) (5log(10)PFU). Mild, transient injection site reactions and flu-like symptoms were noted in all treatment groups, with no significant difference between the groups. Nearly all subjects inoculated with ChimeriVax-JE at both dose levels developed a transient, low-level viremia which was similar in magnitude and duration to that following YF-VAX). Neutralizing antibody seroconversion rates to ChimeriVax-JE was 100% in the high and low dose groups in both na?ve and YF immune subjects; seroconversion to wild-type JE strains was similar or lower than to the homologous (vaccine) virus. Mean neutralizing antibody responses were higher in the ChimeriVax-JE high dose groups (na?ve subjects LNI 1.55, PRNT(50) 254; YF immune subjects LNI 2.23, PRNT(50) 327) than in the low dose groups (na?ve subjects 1.38, PRNT(50) 128; YF immune subjects LNI 1.62, PRNT(50) 270). JE antibody levels were higher in YF immune than in na?ve subjects, dispelling concerns about anti-vector immunity. The safety and immunogenicity profile of ChimeriVax-JE vaccine appears to be similar to that of YF 17D. The new vaccine holds promise for prevention of JE in travelers and residents of endemic countries. The ChimeriVax technology platform is being exploited for development of new vaccines against dengue and West Nile.     

Evaluation of immunity and protective efficacy of a dengue-3 pre-membrane and envelope DNA vaccine in Aotus nancymae monkeys

A dengue (DEN) virus type 3 DNA vaccine expressing pre-membrane and envelope genes was tested for immunogenicity and protective efficacy in Aotus monkeys. Five of six vaccinated animals demonstrated moderate DEN-specific antibody responses as measured by ELISA and virus neutralization in vitro. By contrast, none of the six control animals developed detectable anti-DEN antibodies. When five vaccinated animals were challenged with live DEN-3 virus and viremia determined by PCR amplification of viral RNA in serum samples, one animal was completely protected and two were partially protected as indicated by a decrease in mean days of viremia. The results demonstrate the ability of the DEN-3 DNA vaccine to elicit a neutralizing antibody response and to partially protect against live virus challenge. These findings support the inclusion of this construct in a tetravalent DNA vaccine.     

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

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 na?ve 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.     

冻干水痘减毒活疫苗最小免疫剂量的研究

通过临床研究确定冻干水痘减毒活疫苗的最小免疫剂量 ,为制定规程中的免疫剂量提供科学依据。观察对象接种不同免疫剂量的冻干水痘减毒活疫苗后 ,于接种前和接种后 6周采血 ,采用荧光抗膜抗体 (FAMA)法检测其抗体阳转率和几何平均滴度 (GMT)。接种不同免疫剂量的疫苗 ,抗体阳转率差异无显著的统计学意义。疫苗中抗原含量在 2 5 0 0 0PFU/ml和 2 0 0 0PFU/ml之间 ,GMT差异无显著的统计学意义 ,但 2 5 0 0 0PFU/ml、2 0 0 0PFU/ml与 2 0 0PFU/ml之间抗体GMT的差异有极显著的统计学意义。研究结果表明 ,2 0 0 0PFU/ml为冻干水痘减毒活疫苗的最小免疫剂量。     

Randomized comparative study of the serum antihemagglutinin and antineuraminidase antibody responses to six licensed trivalent influenza vaccines

Background

Serum antibody to the hemagglutinin (HA) surface protein of influenza virus induced by influenza vaccination is a correlate of protection against influenza. The neuraminidase (NA) protein is also on the surface of the virus; antibody to it has been shown to impair virus release from infected cells and to reduce the intensity of influenza infections in animal models and in humans challenged with infectious virus. Recently we have shown that NA inhibiting antibody can independently contribute to immunity to naturally-occurring influenza immunity in the presence of antibody to the HA.

Purpose

The present study was conducted to evaluate induction of antibody to the NA and the HA by commercially available influenza vaccines.

Methods

Healthy young adults were vaccinated with one of five commercially available trivalent inactivated vaccines or live influenza vaccine. Frequencies of serum antibody and fold geometric mean titer (GMT) increases four weeks later were measured to each of the three vaccine viruses (A/H1N1, A/H3N2, B) in hemagglutination-inhibition (HAI) and neutralization (neut) assays. Frequency and fold GMT increase in neuraminidase-inhibition (NI) antibody titers were measured to the influenza A viruses (A/H1N1, A/H3N2).

Results

No significant reactogenicity occurred among the vaccinated subjects. The Fluvirin inactivated vaccine induced more anti-HA antibody responses and a higher fold GMT increase than the other inactivated vaccines but there were no major differences in response frequencies or fold GMT increase among the inactivated vaccines. Both the frequency of antibody increase and fold GMT increase were significantly lower for live vaccine than for any inactivated vaccine in HAI and neut assays for all three vaccine viruses. Afluria inactivated vaccine induced more N1 antibody and Fluarix induced more N2 antibody than the other vaccines but all inactivated vaccines induced serum NI antibody. The live vaccine failed to elicit any NI responses for the N2 NA of A/H3N2 virus and frequencies were low for the N1 of A/H1N1 virus.

Conclusions

Trivalent inactivated influenza vaccines with similar HA dosage induce similar serum anti-HA antibody responses in healthy adults. Current inactivated vaccines all induce serum anti-NA antibody to the N1 and N2 NA proteins but some are better than others for N1 or N2. The live vaccine, Flumist, was a poor inducer of either anti-HA or anti-NA serum antibody compared to inactivated vaccine in the healthy adults. In view of the capacity for contributing to immunity to influenza in humans, developing guidelines for NA content and induction of NA antibody is desirable.     

Vaccination of immunocompetent elderly subjects with a live attenuated Oka strain of varicella zoster virus: a randomized, controlled, dose-response trial

After primary infection in childhood, varicella zoster virus (VZV) remains latent in the dorsal route ganglia. Its reactivation later in life can lead to a zoster episode. VZV-specific, T-cell-mediated immunity (VZV-CMI) is likely to be important in preventing symptomatic reactivation. As CMI declines with age, a vaccine enhancing VZV-CMI might be effective in decreasing the incidence or severity of zoster in elderly subjects. A randomized, double blind controlled trial assessing CMI responses of elderly subjects immunized with a live attenuated, VZV-Oka vaccine was conducted. Two hundred healthy volunteers (55-75 years of age) received either a single injection of the VZV vaccine (PMC), containing 3200 (Oka 3200), 8500 (Oka 8500), or 41,650 (Oka 41650) PFU of live VZV, or a pneumococcus vaccine control group (Pneumo 23((R)). The immune response to VZV was assessed by measuring the T-cell response to VZV antigens, i.e. proliferation (stimulation index, SI), precursor cell frequency (PCF), cytokine secretion, and antibody titers. Six weeks post-vaccination, VZV-specific SI (adjusted mean values) was significantly greater (P<0.0001) in the 3 vaccine groups (with SI=5. 6 for Oka 3200; SI=5.0 for Oka 8500, and SI=7.2 for Oka 41,650) than in the control group (SI=2.9). The increase in PCF was striking, with 72.4, 91.2 and 85.1 precursors per million cells respectively in these 3 vaccine groups, vs 26.3 in the control group. No significant IL-4 secretion was observed in any subject, whereas the presence of IFN-gamma secretion was found to correlate with good responder status. The increase of these CMI parameters did not depend upon the titer of virus injected. Geometric mean titers of VZV antibodies increased in all vaccine groups and remained unchanged in the control group. Nevertheless, no correlation between the antibody response and the cell-mediated response was found. Live attenuated VZV vaccine caused a significant increase in VZV-CMI in a healthy, elderly population. No relationship between vaccine dose and the intensity of the specific response was found.     

A comparison of live and inactivated influenza A (H1N1) virus vaccines. 1. Short-term immunity.

Groups of volunteers were immunized subcutaneously with one of three inactivated influenza virus A/USSR/77 (H1N1) vaccine preparations; a whole virus vaccine, a surface-antigen subunit adsorbed vaccine, or an aqueous surface-antigen subunit vaccine. The reactions to immunization were recorded, and the antibody response was measured 1 month later. A fourth group of volunteers were inoculated intranasally with live attentuated A/USSR/77 (H1N1) influenza virus; the reactions and antibody response of these volunteers were also measured. One month after immunization, the incidence of infection by challenge with homologous live attentuated virus was determined for all groups of volunteers. The results showed that all four vaccines used were relatively non-reactogenic, and that inactivated vaccines induced higher titres of serum antibody than the live attenuated vaccine. All the vaccines induced significant protection against challenge virus infection which was directly related to the level of serum HI antibody response.     

Inactivated yellow fever 17D vaccine: Development and nonclinical safety,immunogenicity and protective activity

In the last 10 years new concerns have arisen about safety of the live, attenuated yellow fever (YF) 17D vaccine, in particular viscerotropic adverse events, which have a case-fatality rate of 64%. A non-replicating cell culture-based vaccine would not cause these adverse events, and potentially could be used in persons with precautions or contraindications to use of the live vaccine, including age <9 months and >60 years, egg allergy, immune suppression, and pregnancy. We developed a whole virion vaccine from the 17D strain inactivated with β-propiolactone, and adsorbed to aluminum hydroxide. The inactivated vaccine was highly immunogenic in mice, hamsters, and cynomolgus macaques. After a single dose in hamsters and macaques, neutralizing antibody titers were similar to those elicited by the live 17D vaccine (YF-VAX^®, Sanofi Pasteur). After two doses of inactivated vaccine, neutralizing antibody titers in hamsters were significantly higher than after a single dose of YF-VAX^® [geometric mean titer (GMT) 20,480 vs. 1940, respectively (P < 0.001, ANOVA)]. Hamsters given a single dose or two doses of inactivated vaccine or a single dose of YF-VAX^® were fully protected against hepatitis, viremia, weight loss and death after challenge with YF virus (Jimenez strain). A clinical trial of the inactivated vaccine (XRX-001) has been initiated.     

A graded-dose study of inactivated, surface antigen influenza B vaccine in volunteers: reactogenicity, antibody response and protection to challenge virus infection.

One hundred and nineteen volunteers were divided into five groups, and each volunteer inoculated subcutaneously with an aqueous subunit B/Hong Kong/73 vaccine containing 40, 20, 10, or 5 micrograms of HA or saline alone in a 0.5 ml volume. The incidence of reactions was recorded 24 h after inoculation. One month following immunization the serum HI antibody to B/Hong Kong/73 virus was measured; each volunteer was inoculated intranasally with live, attenuated influenza B (RB77) virus; and the incidence of infection by the challenge virus was determined by HI antibody response. The results showed that the incidence of reactions to all doses of vaccine were relatively low, the severity mild, and the duration short. However, the incidence of reactions was highest for those given 40 micrograms HA and least for those given 5 micrograms HA. The serum HI antibody responses to vaccine showed a dose-response relationship. For volunteers given 40 micrograms HA, 22 (96%) showed a fourfold rise in antibody titre and all volunteers had antibody titres of greater than 40 following immunization: for volunteers given 5 micrograms HA the g.m.t. increased from 16.6 to 86.1; and for those given 10 and 20 micrograms HA the response was intermediate. Following challenge, the lowest incidence of infection was seen in volunteers given the highest dose of vaccine. However, all doses of vaccine induced some protection against challenge virus infection, and the incidence of infection was directly related to the serum antibody titre at the time of challenge. The 50% protection titre of serum HI antibody was estimated as 15 to 20.     

A graded-dose study of inactivated, surface antigen influenza B vaccine in volunteers: reactogenicity, antibody response and protection to challenge virus infection

One hundred and nineteen volunteers were divided into five groups, and each volunteer inoculated subcutaneously with an aqueous subunit B/Hong Kong/73 vaccine containing 40, 20, 10, or 5 micrograms of HA or saline alone in a 0.5 ml volume. The incidence of reactions was recorded 24 h after inoculation. One month following immunization the serum HI antibody to B/Hong Kong/73 virus was measured; each volunteer was inoculated intranasally with live, attenuated influenza B (RB77) virus; and the incidence of infection by the challenge virus was determined by HI antibody response. The results showed that the incidence of reactions to all doses of vaccine were relatively low, the severity mild, and the duration short. However, the incidence of reactions was highest for those given 40 micrograms HA and least for those given 5 micrograms HA. The serum HI antibody responses to vaccine showed a dose-response relationship. For volunteers given 40 micrograms HA, 22 (96%) showed a fourfold rise in antibody titre and all volunteers had antibody titres of greater than 40 following immunization: for volunteers given 5 micrograms HA the g.m.t. increased from 16.6 to 86.1; and for those given 10 and 20 micrograms HA the response was intermediate. Following challenge, the lowest incidence of infection was seen in volunteers given the highest dose of vaccine. However, all doses of vaccine induced some protection against challenge virus infection, and the incidence of infection was directly related to the serum antibody titre at the time of challenge. The 50% protection titre of serum HI antibody was estimated as 15 to 20.     

Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates

We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 μg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 μg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 μg groups and all but one in the DEN4-80EZip 100 μg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 μg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 μg) and high (50, 50, 50, 100 μg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.