Induction of Th-1 and Th-2 Responses by Respiratory Syncytial Virus Attachment Glycoprotein Is Epitope and Major Histocompatibility Complex Independent

In BALB/c mice, sensitization to respiratory syncytial virus (RSV) attachment (G) glycoprotein leads to the development of lung eosinophilia upon challenge infection with RSV, a pathology indicative of a strong in vivo induction of a Th-2-type response. In this study, we found that a strong, RSV G-specific, Th-1-type cytokine response occurred simultaneously with a Th-2-type response in G-primed mice after RSV challenge. Both Th-1 and Th-2 effector CD4(+) T cells recognized a single immunodominant site on this protein, implying that the differentiation of memory CD4(+) T cells along the Th-1 or Th-2 effector pathway was independent of the epitope specificity of the T cells. A similar observation was made in G-primed H-2(b) haplotype mice after RSV challenge, further suggesting that this process is not dependent on the peptide epitope presented. On the other hand, genes mapping to loci outside of the major histocompatibility complex region are crucial regulators of the development of a Th-2-type response and lung eosinophilia. The implication of these findings for the immune mechanisms underlying the pathogenesis of RSV is discussed.     

Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F_85–93, that carries the RSV CD8⁺ T cell epitope F_85–93 in its neuraminidase stalk. F_85–93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F_85-93 virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F_85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F_85-93 infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8⁺ T cell-mediated immunity by an influenza A virus vector expressing the RSV F_85-93 epitope.     

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2^nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2^nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2^nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.     

A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology

CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against D^bM₁₈₇ epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each D^bM₁₈₇ T effector cell lysed more infected targets on a per cell basis than the numerically dominant K^dM2₈₂ T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized K^dM2₈₂ T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity.     

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na⁺/H⁺ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.     

两种单负链RNA呼吸道病毒——流感病毒和呼吸道合胞病毒感染对粘蛋白1表达调控差异的研究

为进一步了解流感病毒(IFZ)是否像呼吸道合胞病毒(RSV)一样在感染过程中可以上调呼吸道上皮细胞粘蛋白1(MUC1)的表达,进而限制炎症的发展。我们利用qRT-PCR和Western Blot检测两种呼吸道单负链RNA病毒感染人呼吸道上皮细胞系——A549细胞后对MUC1的表达调控。分别利用人喉上皮细胞系——HEp-2细胞系和MDCK细胞系培养并收集RSV和IFZ。相同滴度的两种病毒分别感染A549细胞24h后,分别裂解各组细胞,收集总RNA,应用qRT-PCR检测MUC1mRNA的表达情况;或相同滴度的两种病毒分别感染A549细胞24h和48h后,裂解细胞收集总蛋白,同时收集细胞培养上清,应用Western Blot检测MUC1蛋白的表达情况,应用ELISA检测上清中TNF-α水平。结果显示,虽然两种病毒都能上调TNF-α水平,但只有RSV可以上调呼吸道上皮细胞MUC1表达,并呈剂量效应,而IFZ不能上调呼吸道上皮细胞MUC1的表达。本研究首次探讨两种常见呼吸道单负链RNA病毒感染呼吸道上皮细胞后对MUC1表达调控的差异,初步证实临床上IFZ感染后病情自限性的机制不同于RSV感染,与MUC1的表达上调无关。     

Effect of Complement and Viral Filtration on the Neutralization of Respiratory Syncytial Virus

The addition of 10 hemolytic units of guinea pig complement has been shown to enhance the neutralizing capacity of respiratory syncytial (RS) immune sera produced in guinea pigs and ferrets. This same immune sera, when tested without complement, had little or no neutralizing capacity. The addition of complement to RS immune horse serum did not significantly increase its neutralizing capacity. Immune horse serum effectively neutralized RS virus without complement. Other studies indicated that a 50% tissue culture infective dose of between 30 and 100 should be used in RS serum neutralization tests and that incubation should be for 90 to 105 min at room temperature. The neutralizing capacity of guinea pig immune serum was not increased by the use of filtered virus. The rate of virus neutralization, however, was increased with the addition of 10 hemolytic units of complement. The neutralizing capacity of RS immune horse serum was much greater for filtered than for unfiltered RS virus. The addition of complement increased the rate of virus neutralization but did not increase the neutralizing capacity of the horse immune serum.     

Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability

The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.     

Characterization of Respiratory Syncytial Virus M- and M2-Specific CD4 T Cells in a Murine Model

CD4 T cells have been shown to play an important role in the immunity and immunopathogenesis of respiratory syncytial virus (RSV) infection. We identified two novel CD4 T-cell epitopes in the RSV M and M2 proteins with core sequences M_213-223 (FKYIKPQSQFI) and M2_27-37 (YFEWPPHALLV). Peptides containing the epitopes stimulated RSV-specific CD4 T cells to produce gamma interferon (IFN-γ), interleukin 2 (IL-2), and other Th1- and Th2-type cytokines in an I-A^b-restricted pattern. Construction of fluorochrome-conjugated peptide-I-A^b class II tetramers revealed RSV M- and M2-specific CD4 T-cell responses in RSV-infected mice in a hierarchical pattern. Peptide-activated CD4 T cells from lungs were more activated and differentiated, and had greater IFN-γ expression, than CD4 T cells from the spleen, which, in contrast, produced greater levels of IL-2. In addition, M_209-223 peptide-activated CD4 T cells reduced IFN-γ and IL-2 production in M- and M2-specific CD8 T-cell responses to D^b-M_187-195 and K^d-M2_82-90 peptides more than M2_25-39 peptide-stimulated CD4 T cells. This correlated with the fact that I-A^b-M_209-223 tetramer-positive cells responding to primary RSV infection had a much higher frequency of FoxP3 expression than I-A^b-M2_26-39 tetramer-positive CD4 T cells, suggesting that the M-specific CD4 T-cell response has greater regulatory function. Characterization of epitope-specific CD4 T cells by novel fluorochrome-conjugated peptide-I-A^b tetramers allows detailed analysis of their roles in RSV pathogenesis and immunity.CD4 T lymphocytes play an important role in the resolution of primary viral infections and the prevention of reinfection by regulating a variety of humoral and cellular immune responses. CD4 T cells provide cytokines and other molecules to support the differentiation and expansion of antigen-specific CD8 T cells, which are major effectors for both virus clearance and immunopathology during primary infection with respiratory syncytial virus (RSV) (3, 17, 42, 43). CD4 T-cell help is mandatory for an effective B-cell response (14), which is necessary for producing neutralizing antibodies that prevent secondary RSV infection (12, 18, 21). A concurrent CD4 T-cell response also promotes the maintenance of CD8 T-cell surveillance and effector capacity (9). Previous studies have shown that interleukin 2 (IL-2) from CD4 T cells can restore CD8 T-cell function in lungs (10) and that IL-2 supplementation can increase the production of gamma interferon (IFN-γ) by CD8 T cells upon peptide stimulation in vitro (45).While CD4 T cells are important for providing support to host immunity, they have also been associated with immunopathogenesis by playing a key role in the Th2-biased T-cell response (34, 46), which may be the common mechanism of enhanced lung pathology and other disease syndromes shown in murine studies (2, 16, 17, 19, 35). Earlier studies showed the positive association of formalin-inactivated RSV (FI-RSV) immunization-mediated enhanced illness upon subsequent natural RSV infection with a Th2-biased CD4 T-cell response (19, 44). Th2-orientated CD4 T cells elicit severe pneumonia with extensive eosinophilic infiltrates in the lungs of FI-RSV-immunized mice (13, 24, 48). Patients with severe RSV disease showed an elevated Th2/Th1 cytokine ratio in nasal secretions and peripheral blood mononuclear cells (27, 29, 31, 38). Increased disease severity has also been associated with polymorphisms in Th2-related cytokine genes, such as the IL-4, IL-4 receptor, and IL-13 genes (11, 23, 36). Th2 cytokines from CD4 T cells can also diminish the CD8 T-cell response and delay viral clearance (4, 8).The evaluation of CD4 T-cell responses in viral infection is particularly relevant in the RSV model because of the association of RSV and allergic inflammation, which is largely mediated by CD4 T cells. Understanding the influence of CD4 T cells on CD8 T-cell responses and other immunological effector mechanisms is central to understanding RSV pathogenesis and developing preventive vaccine strategies for RSV. Our lab and others have demonstrated that CD8 T cells target RSV M and M2 proteins with cytolytic effector activities (28, 30, 39). In this study, we found that both RSV M and M2 proteins also contain CD4 T-cell epitopes. These epitopes have 11-mer amino acid core sequences and are associated with the major histocompatibility complex (MHC) class II molecule I-A^b. Fluorochrome-conjugated peptide-I-A^b molecule tetrameric complexes can identify RSV M- and M2-specific CD4 T cells from CB6F1 mice following RSV infection in a hierarchical pattern. Peptides containing the epitopes can stimulate CD4 T cells from RSV M or M2 DNA-immunized and virus-challenged mice and can lead to the production of IFN-γ, IL-2, and other Th1- and Th2-type cytokines that can modulate the CD8 T-cell response to RSV M and M2. We also found that CD4 T cells from the lungs and spleens of immunized mice have different phenotype and cytokine profiles upon in vitro stimulation. These observations suggest a regulatory role for CD4 T cells in the host response to RSV infection. The development of novel MHC class II tetramer reagents allows the characterization of epitope-specific CD4 T-cell responses to RSV and will enable the investigation of basic mechanisms by which CD4 T cells affect pathogenesis and immunity to viral infections.     

T Cell Epitope Mapping of the E-Protein of West Nile Virus in BALB/c Mice

West Nile virus (WNV) is a zoonotic virus, which is transmitted by mosquitoes. It is the causative agent of the disease syndrome called West Nile fever. In some human cases, a WNV infection can be associated with severe neurological symptoms. The immune response to WNV is multifactorial and includes both humoral and cellular immunity. T-cell epitope mapping of the WNV envelope (E) protein has been performed in C57BL/6 mice, but not in BALB/c mice. Therefore, we performed in BALB/c mice a T-cell epitope mapping using a series of peptides spanning the WNV envelope (E) protein. To this end, the WNV-E specific T cell repertoire was first expanded by vaccinating BALB/c mice with a DNA vaccine that generates subviral particles that resemble West Nile virus. Furthermore, the WNV structural protein was expressed in Escherichia coli as a series of overlapping 20-mer peptides fused to a carrier-protein. Cytokine-based ELISPOT assays using these purified peptides revealed positive WNV-specific T cell responses to peptides within the different domains of the E-protein.