A candidate live inactivatable attenuated vaccine for AIDS.

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.     

Identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants

BACKGROUND: Recombination technology can be used to create live attenuated respiratory syncytial virus (RSV) vaccines that contain combinations of known attenuating mutations. METHODS: Two live attenuated, recombinantly derived RSV vaccine candidates, rA2cp248/404 Delta SH and rA2cp248/404/1030 Delta SH, were evaluated in 31 adults and in 95 children >/=6 months old. rA2cp248/404/1030 Delta SH was subsequently evaluated in 44 infants 1-2 months old. These vaccine candidates share 4 attenuating genetic elements and differ only in a missense mutation (1030) in the polymerase gene. RESULTS: Both vaccines were highly attenuated in adults and RSV-seropositive children and were well tolerated and immunogenic in RSV-seronegative children. Compared with that of rA2cp248/404 Delta SH, replication of rA2cp248/404/1030 Delta SH was restricted in RSV-seronegative children (mean peak titer, 10(4.3) vs. 10(2.5) plaque-forming units [pfu]/mL), indicating that the 1030 mutation had a potent attenuating effect. Although rA2cp248/404/1030 Delta SH was well tolerated in infants, only 44% of infants who received two 10(5.3)-pfu doses of vaccine had detectable antibody responses. However, replication after administration of the second dose was highly restricted, indicating that protective immunity was induced. At least 4 of 5 attenuating genetic elements were retained in recovered vaccine viruses. CONCLUSIONS: rA2cp248/404/1030 Delta SH is the first RSV vaccine candidate to be sufficiently attenuated in young infants. Additional studies are needed to determine whether rA2cp248/404/1030 Delta SH can induce protective immunity against wild-type RSV.     

Limited potential for mosquito transmission of a live, attenuated chikungunya virus vaccine.

Studies were conducted to determine the potential for transmission of a live, attenuated chikungunya (CHIK) virus vaccine by orally exposed or virus-inoculated mosquitoes. The vaccine (CHIK 181/clone 25) replicated in and was transmitted by female Aedes albopictus and Ae. aegypti after intrathoracic inoculation. Mosquitoes also became infected with the vaccine after ingesting virus from either a blood-soaked cotton pledget or a viremic monkey. However, because of the low viremias produced in inoculated humans, it is unlikely that mosquitoes would become infected by feeding on a person inoculated with the live, attenuated CHIK vaccine. Although the vaccine was transmitted by mosquitoes after intrathoracic inoculation, there was no evidence of reversion to a virulent phenotype.     

A promoter mutation in the haemagglutinin segment of influenza A virus generates an effective candidate live attenuated vaccine

Background

Annual seasonal and pandemic influenza vaccines need to be produced in a very tight time frame. Haemagglutinin (HA) is the major immunogenic component of influenza vaccines, and there is a lot of interest in improving candidate vaccine viruses.

Objectives

It has been shown elsewhere that mutations introduced in the non-coding region of influenza genome segments can upregulate protein expression. Our objective was to assess a virus based on the laboratory strain A/PR/8/34 (PR8) containing a modified 3′ non-coding region of RNA segment 4 (haemagglutinin).

Methods

NIBRG-93 was generated using reverse genetics. HA protein expression and growth properties were assessed. The virus phenotype suggested that it could be a candidate for use as a live attenuated vaccine, so in vivo studies were performed to assess its suitability.

Results

NIBRG-93 virus has enhanced haemagglutinin production and is significantly attenuated. Electron microscopy (EM) shows that the modified virus produces a large proportion of ‘virus-like particles’ that consist of budded cell membrane covered in HA but lacking M1 protein. The virus was shown to be attenuated in mice and offered complete protection against lethal challenge.

Conclusions

We demonstrate that NIBRG-93 is an effective live attenuated vaccine virus protecting mice against lethal challenge and reducing virus shedding.     

甲型肝炎减毒活疫苗病毒(H2株)人群水平传播的能力

目的观察甲型肝炎（甲肝）减毒活疫苗（H2减毒株）接种后病毒在密切接触者中是否传播.方法分别于1987年5月对浙江省医学科学院职工和家属5例、1988年4月对杭州市某小学2（2）班学生15例及1989年3月对同一所小学1（1）班、1（3）班学生42例,共62例志愿者进行甲肝减毒活疫苗（H2减毒株）接种.采用酶联免疫吸附试验（ELISA）检测接种者血清甲肝IgM抗体及总抗体,采用ELISA双夹心捕捉抗原法和细胞培养法检测5名志愿者接种后的粪便.甲肝的诊断依据临床表现、转氨酶升高和甲肝IgM抗体等标准.结果4例志愿者接种后的31份粪便用ELISA双夹心法检测甲肝抗原全部阴性.取上述其中8份标本接种人胚肺二倍体细胞（KMB17）,经二次传代有3例接种者在8～21 d时检测到甲肝病毒（HAV）.与42例接种者抗体全部阳转不同,在免疫接种后的10～11个月,作为密切接触者的24名同班同学,甲肝抗体阴性.在部分接种甲肝减毒活疫苗的班级中观察了经历一次自然甲肝野毒流行后的免疫反应.50名学生中,15名接种者甲肝抗体全部阳性,未接种者中30名甲肝抗体阴性,5名甲肝IgM抗体阳性.结论甲肝减毒活疫苗（H2减毒株）接种后,有少量HAV疫苗病毒在粪便中排出,但目前未观察到在人群中引起水平传播.     

Safety and immunogenicity of a live attenuated hepatitis A virus vaccine in seronegative volunteers.

Seronegative adults were enrolled in a dose-escalating study of a live attenuated hepatitis A virus (HAV) vaccine that was prepared from the F' variant of HAV strain CR326F. They were injected subcutaneously with 10(4.1), 10(5.2), 10(6.1), or 10(7.3) TCID 50 of HAV vaccine (n = 40) or with placebo (n = 12) and were followed for 6 months. None of the vaccine recipients developed significant systemic reactions or aminotransferase elevations. HAV was not isolated in cell culture from any postvaccination serum or stool specimen tested. Antibody to HAV was detected by modifications of HAV antibody assays (HAVAB or HAVAB-M) in 20%, 40%, 60%, and 100% of the recipients of each vaccine dose, in ascending order. Neutralizing antibody was present in all 10(7.3) TCID50 recipients tested at 3 and 6 months after vaccination. This live attenuated HAV vaccine was well tolerated and highly immunogenic at a dose of 10(7.3) TCID50.     

Dynamics of susceptibility and transmissibility of the live, attenuated, candidate vaccines dengue-1 PDK13, dengue-3 PGMK30F3, and dengue-4 PDK48 after oral infection in Aedes aegypti.

Dengue-1 virus PDK13 and isolates from vaccinees (dengue-1 Ib1 and dengue-1 Ib 10), dengue-3 PGMK30F3, and dengue-4 PDK48 were studied for their abilities to infect, disseminate, and replicate in Aedes aegypti mosquitoes by the oral route. In general, infection and dissemination rates were poorer for the vaccine compared with the parent viruses. The transmissibility was also lower for dengue-1 PDK13 than the parent virus, whereas it was not detected for isolates from vaccinees and dengue-3 PGMK30F3. Transmissibility of dengue-4 PDK48 was not determined because no dissemination occurred. Replication rates of vaccine strains were also found to be less efficient than the parent viruses. These imply that vaccination with the candidate vaccine is safe. Moreover, vector attenuation of vaccine viruses was consistent with its phenotypic markers of attenuation, which remained stable after a mosquito passage or after human and mosquito passage.     

Characterization of antibody responses to combinations of a dengue-2 DNA and dengue-2 recombinant subunit vaccine.

A dengue-2 (DEN-2) DNA vaccine coding for the premembrane and envelope (E) proteins and a recombinant fusion protein containing the B domain of the DEN-2 E protein fused to the maltose-binding protein (MBP) of Escherichia coli both elicited neutralizing antibody in mice. In order to achieve more rapid protective immunity as well as to increase the persistence of neutralizing antibody, we primed mice with the DNA vaccine (D), the recombinant MBP protein (R), or both (RD) given simultaneously, and then boosted twice with either the R (R/R/R or D/R/R) or D (D/D/D or R/D/D) constructs alone or the RD (RD/RD/RD) combination. All of the recombinant protein vaccines were given with alum as an adjuvant. The serum antibody response measured by enzyme-linked immunosorbent assay was highest in D/D/D mice and RD/RD/RD mice. The D/R/R mice showed an intermediate response, and the R/D/D and R/R/R showed the lowest response. The geometric mean (GM) 50% neutralizationtiter (50% plaque reduction neutralization, or PRNT50) was marginally higher for RD/RD/RD mice (891) at 9 months after priming than that for R/R/R mice (724). T he lowest GM PRNT50 titers were seen in the D/D/D mice (33) and R/D/D mice (40), and the D/R/R group had a slightly higher titer (156) than these 2 groups. The predominant antibody subclass for the D/D/D mice was immunoglobulin (Ig) G2a, similar to mice infected with live virus. The R/R/R mice showed an exclusive IgGI antibody response, and the RD/RD/RD response also was predominantly IgGI. The antibody subclass pattern of the R/D/D and D/R/R mice showed a more balanced distribution of both IgG1 and IgG2a. Investigating the neutralizing capacity of antibody subclasses suggested that both IgG1 and IgG2a could neutralize DEN-2 virus. Our observations indicate that the combination RD prime-boost regimen warrants further investigation as a vaccine strategy to prevent dengue infection.     

rDEN4delta30, a live attenuated dengue virus type 4 vaccine candidate, is safe, immunogenic, and highly infectious in healthy adult volunteers

BACKGROUND: The live attenuated dengue virus type 4 (DEN-4) vaccine candidate virus rDEN4 Delta 30 was previously found to be safe and immunogenic at a dose of 10(5) plaque-forming units (pfu). METHODS: In a follow-up placebo-controlled phase 2 clinical trial, rDEN4 Delta 30 was administered as a single inoculation to 3 separate dose cohorts (10(3) pfu, 10(2) pfu, or 10(1) pfu), for further evaluation. Each dose cohort consisted of 20 vaccinees and 4 placebo recipients. Volunteers were monitored closely for adverse events, and serum was collected on study days 28 and 42 for determination of neutralizing antibody titer. RESULTS: The vaccine was well tolerated at all doses studied. The most common adverse events observed were a transient asymptomatic rash in >50% of vaccinees and a mild neutropenia in approximately 20% of vaccinees. No vaccinee developed a dengue-like illness. The vaccine was highly infectious and immunogenic, with 95%-100% of vaccinees in each dose cohort developing a >/=4-fold increase in titers of serum neutralizing antibodies against DEN-4. CONCLUSIONS: The rDEN4 Delta 30 vaccine is safe and induced an antibody response that was broadly neutralizing against genotypically diverse DEN-4 viruses. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation.     

Phase I clinical evaluation of rDEN4Delta30-200,201: a live attenuated dengue 4 vaccine candidate designed for decreased hepatotoxicity

The rDEN4Delta30-200,201 is a live attenuated DENV-4 vaccine candidate specifically designed to further attenuate the rDEN4Delta30 parent virus. In the present study, 28 healthy adult volunteers were randomized to receive either 10(5) plaque-forming unit (PFU) of vaccine (20) or placebo (8) as a single subcutaneous injection. Volunteers were evaluated for safety every other day for 16 days. Serum neutralizing antibody titer against DEN4 was determined at study day 28, 42, and 180. The vaccine infected all vaccinees and was well tolerated without inducing alanine aminotransferase (ALT) elevations. Although virus was not recovered from the serum of any vaccinee, moderate levels of neutralizing antibody were induced in all volunteers. Thus the restricted replication of rDEN4Delta30-200,201 previously documented in animal models was confirmed in humans. The rDEN4Delta30-200,201 is a promising candidate and can be considered for inclusion in a tetravalent dengue virus (DENV) vaccine.     

Molecular epidemiology of live, attenuated varicella virus vaccine in children with leukemia and in normal adults

Restriction endonuclease analysis of varicella-zoster virus (VZV) DNA has been used in unraveling the complex epidemiology of VZV infections in individuals immunized with a live, attenuated varicella virus vaccine. Early rashes appearing within the first six weeks after vaccination are invariably due to vaccine virus. True breakthrough infections with wild-type VZV also occur in vaccinees. Five cases of zoster have been seen in leukemic children vaccinated while in remission. One case appeared 22 months after vaccination in the same general area as the inoculation. The virus isolated was vaccine derived. A second case of zoster appeared in a dermatome unrelated to the sites of vaccination approximately 19 months after apparently natural varicella. This virus was wild type. Vaccine virus can therefore establish latency and can later reactivate as herpes zoster.     

Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region.

The recombinant dengue virus type-4 vaccine candidate 2AA30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3'-untranslated region of the viral genome. A clinical trial to evaluate the safety and immunogenicity of a single dose of 2Adelta30 was conducted with 20 adult human volunteers. The vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. Viremia was detectable in 14 volunteers at a mean level of 1.6 log10 plaque-forming units/ml of serum, although all 20 volunteers seroconverted with a seven-fold or greater increase in serum neutralizing antibody titer on day 28 post-vaccination (mean titer = 1:580). A mild, asymptomatic, macular rash developed in 10 volunteers, and a transient elevation in the serum level of alanine aminotransferase was noted in five volunteers. The low level of reactogenicity and high degree of immunogenicity of this vaccine candidate warrant its further evaluation and its use to create chimeric vaccine viruses expressing the structural genes of dengue virus types 1, 2, and 3.     

Limited potential for transmission of live dengue virus vaccine candidates by Aedes aegypti and Aedes albopictus

To evaluate the transmission risk of four live dengue (DEN) vaccine candidates developed by the U.S. Army (DEN-1, 45AZ5 PDK 20; DEN-2, S16803 PDK 50; DEN-3, CH53489 PDK 20; and DEN-4, 341750 PDK 20), we tested 3,010 Aedes aegypti and 1,576 Aedes albopictus mosquitoes blood-fed on 21 volunteers who had been administered one of the four vaccine candidates or the licensed yellow fever (YF) vaccine (17D). We used an indirect immunofluorescence assay (IFA) to detect DEN or YF viral antigen in the heads of mosquitoes. Corresponding to the lack of a detectable viremia among volunteers inoculated 8-13 days previously with live DEN-1 or DEN-2 vaccine candidates, only six mosquitoes developed disseminated infections after feeding on these volunteers. These six mosquitoes included 4 of 247 Ae. albopictus fed on volunteers inoculated with the DEN-1 vaccine candidate and 2 of 528 Ae. aegypti fed on volunteers inoculated with the DEN-2 vaccine candidate. Infection was confirmed in each of these IFA-positive mosquitoes by isolating infectious virus from the mosquito's body in Vero-cell culture. None of the 1,252 or the 969 mosquitoes fed on DEN-3 or DEN-4 recipients, respectively, were infected. Overall, dissemination rates in Ae. albopictus and Ae. aegypti were low. Dissemination rates were 0.5%, 0.3%, < 0.1%, and < 0.1% for the DEN-1 through DEN-4 vaccine candidates, respectively. Because of the observed low dissemination rates, it is unlikely that these vaccine viruses would be transmitted under natural conditions.     

Immunity to whole varicella-zoster virus antigen and glycoproteins I and p170: relation to the immunizing regimen of live attenuated varicella vaccine

Humoral and cellular immune responses to whole varicella-zoster virus (VZV) antigen and the VZV proteins glycoprotein I (gpI) and nonglycosylated protein p170 were evaluated in healthy children and adults given lyophilized live-attenuated varicella vaccine. Children received one dose of vaccine containing 950 pfu, whereas adults received two doses of 2500 pfu. After one year, the antibody titers of adult vaccinees to whole VZV and to gpI were significantly higher than those of children. Antibody titers to whole VZV, gpI, and p170 were lower among both vaccine populations than titers in naturally immune individuals, but vaccinees who seroconverted initially retained detectable VZV antibodies. Using T lymphocyte proliferation to measure cellular immunity, we found the mean (+/- SE) transformation index to whole VZV antigen to be 4.1 +/- 0.96 in children tested at one year, a mean significantly lower than the mean of 12.7 +/- 3.39 in adults and 13.0 +/- 1.67 in naturally immune subjects. These observations suggest that the vaccine dose affects vaccine-induced immunity to VZV.     

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. II. Safety and immunogenicity in humans

To determine safety and immunogenicity, a single 0.5 ml dose of a monovalent live-attenuated dengue (DEN) 4 (341750 Carib) vaccine was given sc to 3 groups of flavivirus nonimmune volunteers in increasing concentrations. Two recipients received 10(3) plaque forming units (PFU)/dose (1:100 dilution of stock vaccine). One remained asymptomatic, but became viremic between days 12 and 15, experienced a mild elevation of temperature (37.4 degrees C), and developed DEN-4 specific antibody. Neither recipient of the 10(4) PFU became infected. Eight volunteers then received undiluted vaccine (10(5) PFU). Viremia and antibody (neutralizing, hemagglutination inhibition, and IgM) developed in 5 of the 8 (63%). These 5 volunteers also developed a scarcely noticeable macular, blanching rash and minimal temperature elevations (37.3, 38.1, 37, 37.9, and 37.9 degrees C). Clinically insignificant decreases in total white blood cell, lymphocyte, and polymorphonuclear cell counts and an elevation in mononuclear cell counts occurred in association with viremia. This vaccine is safe, reasonably immunogenic, and suitable for further evaluation.     

Current status of live attenuated influenza vaccine in the United States for seasonal and pandemic influenza

Abstract A live attenuated influenza vaccine (LAIV) is currently approved in the United States for the prevention of influenza in individuals 2–49 years of age. This article summarizes the available data describing the safety and efficacy of LAIV for the prevention of influenza in both children and adults. LAIV is administered as an intranasal spray and has been shown to provide high levels of efficacy against influenza illness caused by both matched and mismatched strains in children and adults. In studies comparing LAIV and inactivated influenza vaccine in children, LAIV recipients experienced 35–53% fewer cases of culture‐confirmed influenza illness caused by antigenically matched strains. Protection through a second influenza season against antigenically matched strains has also been seen in children. In adults, definitive comparative studies of LAIV and inactivated vaccine have not been conducted and no statistically significant differences in efficacy have been demonstrated. The most common adverse reactions with LAIV include runny nose/nasal congestion in all age groups, fever >100°F in children, and sore throat in adults. Formulations of LAIV against pandemic influenza strains, including H5N1, H9N2, and H7N3, are currently being tested in preclinical and phase I clinical studies.     

Cross-reactive H1N1 antibody responses to a live attenuated influenza vaccine in children: implication for selection of vaccine strains

Cross-reactive antibody responses of 3 trivalent, live attenuated intranasal influenza vaccine (FluMist) formulations containing 3 different H1N1 strains (A/Texas/36/91, A/Shenzhen/227/95, and A/Beijing/262/95) were evaluated in initially seronegative children. FluMist containing A/Shenzhen/227/95 was more likely to induce cross-reactive hemagglutination inhibition (HAI) antibody against A/Texas/36/91 than against A/Beijing/262/95, and FluMist containing A/Beijing/262/95 induced low levels of cross-reactive HAI antibody against A/Shenzhen/227/95 and A/New Caledonia/20/99. The observed differences in HAI cross-reactivity seem to be partly related to the number of amino acid (aa) differences on the hemagglutinin 1 domain (328 aa residues) rather than the hemagglutinin protein (550 aa residues).     

Randomized, controlled study of the safety and immunogenicity of Peru-15, a live attenuated oral vaccine candidate for cholera, in adult volunteers in Bangladesh

BACKGROUND. A live oral Vibrio cholerae O1 El Tor vaccine candidate, Peru-15, was studied for safety, immunogenicity, and excretion in phase 1 (inpatient) and phase 2 (outpatient) studies of Bangladeshi adults.METHODs. The study was conducted among adults, by use of a double-blind, randomized, placebo-controlled design. A single dose of Peru-15 (approximately 2 x 108 cfu) or placebo (buffer only) was given in standard bicarbonate and ascorbic acid buffer.RESULTS. Study treatment did not elicit any major adverse events in the volunteers, during either the inpatient or the outpatient phases, and there were no reports of diarrhea. V. cholerae was isolated from the stool of only 1 volunteer and was found to be genetically identical to the vaccine strain. Vibriocidal antibody responses were seen in 30 (75%) of 40 vaccine recipients and in 3 (10%) of 30 placebo recipients. Peripheral blood immunoglobulin (Ig) A and IgM antibody-secreting cell responses to lipopolysaccharide were seen in the majority of vaccine recipients (response rate, 78%--88%). Seroconversion for lipopolysaccharide-specific IgA antibodies was seen in 88% of vaccine recipients. The response in vaccine recipients was significantly higher than that in placebo recipients, in all of the immunological assays (P=.036 to <.001). A lower immunological response against cholera toxin B subunit was detected.CONCLUSIONS. The safety and immunogenicity of this Peru-15 vaccine candidate indicates the usefulness of future studies in Bangladesh, where cholera is endemic.     

Superiority of live attenuated compared with inactivated influenza A virus vaccines in older, chronically ill adults

Forty-eight older adults with chronic diseases were vaccinated intranasally with live attenuated influenza A/Korea/1/82 (H3N2), CR59 virus. Forty-two (88 percent) CR59 virus recipients became infected with vaccine virus without adverse effects or change in mean pulmonary function even among the 29 infected recipients with moderate to severe chronic obstructive pulmonary disease. Among control groups who received either monovalent or trivalent inactivated influenza virus vaccines intramuscularly, the rates of fourfold rises in serum antibody titer to hemagglutinin (HA) were not different from the rate following CR59 virus inoculation. However, CR59 virus was superior to inactivated virus vaccine at stimulating secretory antibody to HA. Vaccinees age 65 years and older were more likely to shed CR59 virus in nasal secretions than were younger vaccinees, but antibody responses were not different. CR59 virus vaccine was safe and immunogenic in this population and more often induced a nasal wash IgA antibody response than the inactivated virus vaccines.