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Kanta Subbarao

Publications and source records attributed to Kanta Subbarao.

At least 37 records · Page 2Linked to original sources
PubMed2005

Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants.

In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.

Amino Acid Substitution↗
PubMed2005

Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans.

Advanced age has repeatedly been identified as an independent correlate of adverse outcome and a predictor of mortality in cases of severe acute respiratory syndrome (SARS). SARS-associated mortality may exceed 50% for persons aged 60 years or older. Heightened susceptibility of the elderly to severe SARS and the ability of SARS coronavirus to replicate in mice led us to examine whether aged mice might be susceptible to disease. We report here that viral replication in aged mice was associated with clinical illness and pneumonia, demonstrating an age-related susceptibility to SARS disease in animals that parallels the human experience.

Age Factors↗
PubMed2005

Human infections with avian influenza viruses.

Avian influenza A viruses are zoonotic agents that can cause outbreaks of acute respiratory disease in animals and man. The unprecedented re-emergence of highly pathogenic H5N1 influenza viruses in the human population has resulted in the deaths of 42 people and slaughter of millions of birds in Asia. The most striking features of this virus are its ability to infect and cause disease in a wide range of animals and to spread from person to person. There are also instances of infections with H7 and H9 subtypes in humans that are not discussed in this article. Therefore it is crucial to develop virologic surveillance programs for animal and human influenza, to limit the spread of the virus from infected poultry, and to develop efficacious vaccines by exploiting novel technologies such as reverse genetics.

Adult↗
PubMed2004

Replication of SARS coronavirus administered into the respiratory tract of African Green, rhesus and cynomolgus monkeys.

SARS coronavirus (SARS-CoV) administered intranasally and intratracheally to rhesus, cynomolgus and African Green monkeys (AGM) replicated in the respiratory tract but did not induce illness. The titer of serum neutralizing antibodies correlated with the level of virus replication in the respiratory tract (AGM>cynomolgus>rhesus). Moderate to high titers of SARS-CoV with associated interstitial pneumonitis were detected in the lungs of AGMs on day 2 and were resolving by day 4 post-infection. Following challenge of AGMs 2 months later, virus replication was highly restricted and there was no evidence of enhanced disease. These species will be useful for the evaluation of the immunogenicity of candidate vaccines, but the lack of apparent clinical illness in all three species, variability from animal to animal in level of viral replication, and rapid clearance of virus and pneumonitis in AGMs must be taken into account by investigators considering the use of these species in efficacy and challenge studies.

Animals↗
PubMed2004

Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice.

We describe a model of severe acute respiratory syndrome-coronavirus (SARS-CoV) infection in C57BL/6 mice. A clinical isolate of the virus introduced intranasally replicated transiently to high levels in the lungs of these mice, with a peak on day 3 and clearance by day 9 postinfection. Viral RNA localized to bronchial and bronchiolar epithelium. Expression of mRNA for angiotensin converting enzyme 2, the SARS-CoV receptor, was detected in the lung following infection. The virus induced production in the lung of the proinflammatory chemokines CCL2, CCL3, CCL5, CXCL9, and CXCL10 with differential kinetics. The receptors for these chemokines were also detected. Most impressively, mRNA for CXCR3, the receptor for CXCL9 and CXCL10, was massively up-regulated in the lungs of SARS-CoV-infected mice. Surprisingly Th1 (and Th2) cytokines were not detectable, and there was little local accumulation of leukocytes and no obvious clinical signs of pulmonary dysfunction. Moreover, beige, CD1-/-, and RAG1-/- mice cleared the virus normally. Infection spread to the brain as it was cleared from the lung, again without leukocyte accumulation. Infected mice had a relative failure to thrive, gaining weight significantly more slowly than uninfected mice. These data indicate that C57BL/6 mice support transient nonfatal systemic infection with SARS-CoV in the lung, which is able to disseminate to brain. In this species, proinflammatory chemokines may coordinate a rapid and highly effective innate antiviral response in the lung, but NK cells and adaptive cellular immunity are not required for viral clearance.

Administration, Intranasal↗
PubMed2004

An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.

Passive serotherapy can confer immediate protection against microbial infection, but methods to rapidly generate human neutralizing monoclonal antibodies are not yet available. We have developed an improved method for Epstein-Barr virus transformation of human B cells. We used this method to analyze the memory repertoire of a patient who recovered from severe acute respiratory syndrome coronavirus (SARS-CoV) infection and to isolate monoclonal antibodies specific for different viral proteins, including 35 antibodies with in vitro neutralizing activity ranging from 10(-8)M to 10(-11)M. One such antibody confers protection in vivo in a mouse model of SARS-CoV infection. These results show that it is possible to interrogate the memory repertoire of immune donors to rapidly and efficiently isolate neutralizing antibodies that have been selected in the course of natural infection.

Adult↗
PubMed2004

Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS.

BACKGROUND: The outbreak of severe acute respiratory syndrome (SARS) in 2002 was caused by a previously unknown coronavirus-SARS coronavirus (SARS-CoV). We have developed an experimental SARS vaccine for direct immunisation of the respiratory tract, the major site of SARS- coronavirus transmission and disease. METHODS: We expressed the complete SARS coronavirus envelope spike (S) protein from a recombinant attenuated parainfluenza virus (BHPIV3) that is being developed as a live attenuated, intranasal paediatric vaccine against human parainfluenza virus type 3 (HPIV3). We immunised eight African green monkeys, four with a single dose of BHPIV3/ SARS-S and four with a control, BHPIV3/Ctrl, administered via the respiratory tract. A SARS-coronavirus challenge was given to all monkeys 28 days after immunisation. FINDINGS: Immunisation of animals with BHPIV3/SARS-S induced the production of SARS-coronavirus-neutralising serum antibodies, indicating that a systemic immune response resulted from mucosal immunisation. After challenge with SARS coronavirus, all monkeys in the control group shed SARS coronavirus, with shedding lasting 5-8 days. No viral shedding occurred in the group immunised with BHPIV3/SARS-S. INTERPRETATION: A vectored mucosal vaccine expressing the SARS-coronavirus S protein alone may be highly effective in a single-dose format for the prevention of SARS.

Administration, Intranasal↗
PubMed2004

Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity.

We investigated the contributions of the structural proteins of severe acute respiratory syndrome (SARS) coronavirus (CoV) to protective immunity by expressing them individually and in combinations from a recombinant parainfluenza virus (PIV) type 3 vector called BHPIV3. This vector provided direct immunization of the respiratory tract, the major site of SARS transmission, replication, and disease. The BHPIV3/SARS recombinants were evaluated for immunogenicity and protective efficacy in hamsters, which support a high level of pulmonary SARS-CoV replication. A single intranasal administration of BHPIV3 expressing the SARS-CoV spike protein (S) induced a high titer of SARS-CoV-neutralizing serum antibodies, only 2-fold less than that induced by SARS-CoV infection. The expression of S with the two other putative virion envelope proteins, the matrix M and small envelope E proteins, did not augment the neutralizing antibody response. In absence of S, expression of M and E or the nucleocapsid protein N did not induce a detectable serum SARS-CoV-neutralizing antibody response. Immunization with BHPIV3 expressing S provided complete protection against SARS-CoV challenge in the lower respiratory tract and partial protection in the upper respiratory tract. This was augmented slightly by coexpression with M and E. Expression of M, E, or N in the absence of S did not confer detectable protection. These results identify S among the structural proteins as the only significant SARS-CoV neutralization antigen and protective antigen and show that a single mucosal immunization is highly protective in an experimental animal that supports efficient replication of SARS-CoV.

Administration, Intranasal↗
PubMed2004

Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice.

The spike protein (S), a membrane component of severe acute respiratory syndrome coronavirus (SARS-CoV) is anticipated to be an important component of candidate vaccines. We constructed recombinant forms of the highly attenuated modified vaccinia virus Ankara (MVA) containing the gene encoding full-length SARS-CoV S with and without a C-terminal epitope tag called MVA/S-HA and MVA/S, respectively. Cells infected with MVA/Sor MVA/S-HA synthesized a 200-kDa protein, which was recognized by antibody raised against a synthetic peptide of SARS-CoV S or the epitope tag in Western blot analyses. Further studies indicated that S was N-glycosylated and migrated in SDS polyacrylamide gels with an apparent mass of approximately 160 kDa after treatment with peptide N-glycosidase F. The acquisition of resistance to endoglycosidase H indicated trafficking of S to the medial Golgi compartment, and confocal microscopy showed that S was transported to the cell surface. Intranasal or intramuscular inoculations of BALB/c mice with MVA/S produced serum antibodies that recognized the SARS S in ELISA and neutralized SARS-CoV in vitro. Moreover, MVA/S administered by either route elicited protective immunity, as shown by reduced titers of SARS-CoV in the upper and lower respiratory tracts of mice after challenge. Passive transfer of serum from mice immunized with MVA/S to naïve mice also reduced the replication of SARS-CoV in the respiratory tract after challenge, demonstrating a role for antibody to S in protection. The attenuated nature of MVA and the ability of MVA/S to induce neutralizing antibody that protects mice support further development of this candidate vaccine.

Animals↗
PubMed2004

A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice.

Public health measures have successfully identified and contained outbreaks of the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), but concerns remain over the possibility of future recurrences. Finding a vaccine for this virus therefore remains a high priority. Here, we show that a DNA vaccine encoding the spike (S) glycoprotein of the SARS-CoV induces T cell and neutralizing antibody responses, as well as protective immunity, in a mouse model. Alternative forms of S were analysed by DNA immunization. These expression vectors induced robust immune responses mediated by CD4 and CD8 cells, as well as significant antibody titres, measured by enzyme-linked immunosorbent assay. Moreover, antibody responses in mice vaccinated with an expression vector encoding a form of S that includes its transmembrane domain elicited neutralizing antibodies. Viral replication was reduced by more than six orders of magnitude in the lungs of mice vaccinated with these S plasmid DNA expression vectors, and protection was mediated by a humoral but not a T-cell-dependent immune mechanism. Gene-based vaccination for the SARS-CoV elicits effective immune responses that generate protective immunity in an animal model.

Adoptive Transfer↗
PubMed2004

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV.

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment laboratories at biosafety level 3 (BSL3). The virus was inactivated by ultraviolet light (UV) at 254 nm, heat treatment of 65 degrees C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV containing materials, that are rendered non-infectious, to be conducted at reduced safety levels.

Animals↗
PubMed2004

Reassortment and evolution of current human influenza A and B viruses.

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.

Evolution, Molecular↗
PubMed2004

pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN.

The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.

Animals↗
PubMed2004

Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins.

The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nsp1, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nsp14, nsp15, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nsp12 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis.

Animals↗
PubMed2004

Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice.

Following intranasal administration, the severe acute respiratory syndrome (SARS) coronavirus replicated to high titers in the respiratory tracts of BALB/c mice. Peak replication was seen in the absence of disease on day 1 or 2, depending on the dose administered, and the virus was cleared within a week. Viral antigen and nucleic acid were detected in bronchiolar epithelial cells during peak viral replication. Mice developed a neutralizing antibody response and were protected from reinfection 28 days following primary infection. Passive transfer of immune serum to naïve mice prevented virus replication in the lower respiratory tract following intranasal challenge. Thus, antibodies, acting alone, can prevent replication of the SARS coronavirus in the lung, a promising observation for the development of vaccines, immunotherapy, and immunoprophylaxis regimens.

Animals↗
PubMed2003

Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate.

H9N2 subtype avian influenza viruses (AIVs) are widely distributed in avian species and were isolated from humans in Hong Kong and Guangdong province, China in 1999 raising concern of their potential for pandemic spread. We generated a high-growth reassortant virus (G9/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from the H9N2 avian influenza virus A/chicken/Hong Kong/G9/97 (G9) and six internal genes from A/Puerto Rico/8/34 (PR8) by genetic reassortment, for evaluation as a potential vaccine candidate in humans. Pathogenicity studies showed that the G9/PR8 reassortant was not highly pathogenic for mice or chickens. Two doses of a formalin-inactivated G9/PR8 virus vaccine induced hemagglutination inhibiting antibodies and conferred complete protection against challenge with G9 and the antigenically distinct H9N2 A/Hong Kong/1073/99 (G1-like) virus in a mouse model. These results indicate that the high growth G9/PR8 reassortant has properties that are desirable in a vaccine seed virus and is suitable for evaluation in humans for use in the event of an H9 pandemic.

Animals↗
PubMed2003

Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine.

Avian H5N1 influenza viruses isolated from humans in Hong Kong in 1997 were divided into two antigenic groups based on the presence or absence of a potential glycosylation site at amino acid residues 154-156 in the HA1 region of the viral hemagglutinin (HA) surface glycoprotein. To assess the impact of glycosylation on the immunogenicity of an HA-expressing DNA vaccine, a series of plasmid vaccine constructs that differed in the presence of potential glycosylation sites at amino acid residues 154-156, 165-167, and 286-288 were used to immunize BALB/c mice. Postvaccination serum IgG, hemagglutination inhibition, and neutralizing antibody titers as well as the morbidity and mortality following a lethal H5N1 viral challenge did not vary significantly among any of the experimental groups. We conclude that the glycosylation pattern of the influenza virus HA1 domain has little impact on the murine antibody response raised to a DNA vaccine encoding the H5 HA, thereby minimizing the concern that the pattern of glycosylation sites encoded by the vaccine match those of closely related H5 viruses.

Animals↗