Comparative pathogenesis of three human and zoonotic SARS-CoV strains in cynomolgus macaques

The severe acute respiratory syndrome (SARS) epidemic was characterized by increased pathogenicity in the elderly due to an early exacerbated innate host response. SARS-CoV is a zoonotic pathogen that entered the human population through an intermediate host like the palm civet. To prevent future introductions of zoonotic SARS-CoV strains and subsequent transmission into the human population, heterologous disease models are needed to test the efficacy of vaccines and therapeutics against both late human and zoonotic isolates. Here we show that both human and zoonotic SARS-CoV strains can infect cynomolgus macaques and resulted in radiological as well as histopathological changes similar to those seen in mild human cases. Viral replication was higher in animals infected with a late human phase isolate compared to a zoonotic isolate. While there were significant differences in the number of host genes differentially regulated during the host responses between the three SARS-CoV strains, the top pathways and functions were similar and only apparent early during infection with the majority of genes associated with interferon signaling pathways. This study characterizes critical disease models in the evaluation and licensure of therapeutic strategies against SARS-CoV for human use.     

Structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, and detailed phylogenetic and epidemiological analyses have suggested that it originated from animals. The spike (S) glycoprotein has been identified as a major component of protective immunity, and 23 different amino acid changes were noted during the expanding epidemic. Using a panel of SARS-CoV recombinants bearing the S glycoproteins from isolates representing the zoonotic and human early, middle, and late phases of the epidemic, we identified 23 monoclonal antibodies (MAbs) with neutralizing activity against one or multiple SARS-CoV spike variants and determined the presence of at least six distinct neutralizing profiles in the SARS-CoV S glycoprotein. Four of these MAbs showed cross-neutralizing activity against all human and zoonotic S variants in vitro, and at least three of these were mapped in distinct epitopes using escape mutants, structure analyses, and competition assays. These three MAbs (S109.8, S227.14, and S230.15) were tested for use in passive vaccination studies using lethal SARS-CoV challenge models for young and senescent mice with four different homologous and heterologous SARS-CoV S variants. Both S227.14 and S230.15 completely protected young and old mice from weight loss and virus replication in the lungs for all viruses tested, while S109.8 completely protected mice from weight loss and clinical signs in the presence of viral titers. We conclude that a single human MAb can confer broad protection against lethal challenge with multiple zoonotic and human SARS-CoV isolates, and we identify a robust cocktail formulation that targets distinct epitopes and minimizes the likely generation of escape mutants.     

Natural mutations in the receptor binding domain of spike glycoprotein determine the reactivity of cross-neutralization between palm civet coronavirus and severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS) outbreak of 2002 and 2003 occurred as a result of zoonotic transmission. Coronavirus (CoV) found in naturally infected palm civet (civet-CoV) represents the closest genetic relative to SARS-CoV, but the degree and the determinants of cross-neutralization among these viruses remain to be investigated. Studies indicate that the receptor binding domain (RBD) of the SARS-CoV spike (S) glycoprotein contains major determinants for viral entry and neutralization. We aim to characterize the impact of natural mutations within the RBDs of civet-CoVs on viral entry and cross-neutralization. In this study, the S glycoprotein genes were recovered from naturally infected civets in central China (Hubei province), extending the geographic distribution of civet-CoV beyond the southeastern province of Guangdong. Moreover, pseudoviruses generated in our laboratory with four civet S genes, each with a distinct RBD, infected cells expressing human receptor angiotensin-converting enzyme 2, but with 90 to 95% less efficiency compared to that of SARS-CoV. These four civet S genes were also constructed as DNA vaccines to immunize mice. Immunized sera elicited against most civet S glycoproteins displayed potent neutralizing activities against autologous viruses but were much less efficient (50% inhibitory concentration, 20- to 40-fold) at neutralizing SARS-CoV and vice versa. Convalescence-phase sera from humans were similarly ineffective against the dominant civet pseudovirus. Our findings suggest that the design of SARS vaccine should consider not only preventing the reemergence of SARS-CoV but also providing cross-protection, thus interrupting zoonotic transmission of a group of genetically divergent civet CoVs of broad geographic origin.     

Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium

In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and caused over 8,000 human cases of infection and more than 700 deaths worldwide. Zoonotic SARS-CoV likely evolved to infect humans by a series of transmission events between humans and animals for sale in China. Using synthetic biology, we engineered the spike protein (S) from a civet strain, SZ16, into our epidemic strain infectious clone, creating the chimeric virus icSZ16-S, which was infectious but yielded progeny viruses incapable of propagating in vitro. After introducing a K479N mutation within the S receptor binding domain (RBD) of SZ16, the recombinant virus (icSZ16-S K479N) replicated in Vero cells but was severely debilitated in growth. The in vitro evolution of icSZ16-S K479N on human airway epithelial (HAE) cells produced two viruses (icSZ16-S K479N D8 and D22) with enhanced growth on HAE cells and on delayed brain tumor cells expressing the SARS-CoV receptor, human angiotensin I converting enzyme 2 (hACE2). The icSZ16-S K479N D8 and D22 virus RBDs contained mutations in ACE2 contact residues, Y442F and L472F, that remodeled S interactions with hACE2. Further, these viruses were neutralized by a human monoclonal antibody (MAb), S230.15, but the parent icSZ16-S K479N strain was eight times more resistant than the mutants. These data suggest that the human adaptation of zoonotic SARS-CoV strains may select for some variants that are highly susceptible to select MAbs that bind to RBDs. The epidemic, icSZ16-S K479N, and icSZ16-S K479N D22 viruses replicate similarly in the BALB/c mouse lung, highlighting the potential use of these zoonotic spike SARS-CoVs to assess vaccine or serotherapy efficacy in vivo.     

Pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus

Zoonotic severe acute respiratory syndrome coronavirus (SARS-CoV) likely evolved to infect humans by a series of transmission events between humans and animals in markets in China. Virus sequence data suggest that the palm civet served as an amplification host in which civet and human interaction fostered the evolution of the epidemic SARS Urbani strain. The prototypic civet strain of SARS-CoV, SZ16, was isolated from a palm civet but has not been successfully cultured in vitro. To propagate a chimeric recombinant SARS-CoV bearing an SZ16 spike (S) glycoprotein (icSZ16-S), we constructed cell lines expressing the civet ortholog (DBT-cACE2) of the SARS-CoV receptor (hACE2). Zoonotic SARS-CoV was completely dependent on ACE2 for entry. Urbani grew with similar kinetics in both the DBT-cACE2 and the DBT-hACE2 cells, while icSZ16-S only grew in DBT-cACE2 cells. The SZ16-S mutant viruses adapted to human airway epithelial cells and displayed enhanced affinity for hACE2 but exhibited severe growth defects in the DBT-cACE2 cells, suggesting that the evolutionary pathway that promoted efficient hACE2 interactions simultaneously abolished efficient cACE2 interactions. Structural modeling predicted two distinct biochemical interaction networks by which zoonotic receptor binding domain architecture can productively engage hACE2, but only the Urbani mutational repertoire promoted efficient usage of both hACE2 and cACE2 binding interfaces. Since dual species tropism was preserved in Urbani, it is likely that the virus evolved a high affinity for cACE2/hACE2 receptors through adaptation via repeated passages between human and civet hosts. Furthermore, zoonotic SARS-CoV was variably neutralized by antibodies that were effective against the epidemic strain, highlighting their utility for evaluating passive immunization efficacy.     

Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2

Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002-2003 SARS outbreak and during the much less severe 2003-2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002-2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002-2003 SARS epidemic.     

Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections

It is believed that a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), was passed from palm civets to humans and caused the epidemic of SARS in 2002 to 2003. The major species barriers between humans and civets for SARS-CoV infections are the specific interactions between a defined receptor-binding domain (RBD) on a viral spike protein and its host receptor, angiotensin-converting enzyme 2 (ACE2). In this study a chimeric ACE2 bearing the critical N-terminal helix from civet and the remaining peptidase domain from human was constructed, and it was shown that this construct has the same receptor activity as civet ACE2. In addition, crystal structures of the chimeric ACE2 complexed with RBDs from various human and civet SARS-CoV strains were determined. These structures, combined with a previously determined structure of human ACE2 complexed with the RBD from a human SARS-CoV strain, have revealed a structural basis for understanding the major species barriers between humans and civets for SARS-CoV infections. They show that the major species barriers are determined by interactions between four ACE2 residues (residues 31, 35, 38, and 353) and two RBD residues (residues 479 and 487), that early civet SARS-CoV isolates were prevented from infecting human cells due to imbalanced salt bridges at the hydrophobic virus/receptor interface, and that SARS-CoV has evolved to gain sustained infectivity for human cells by eliminating unfavorable free charges at the interface through stepwise mutations at positions 479 and 487. These results enhance our understanding of host adaptations and cross-species infections of SARS-CoV and other emerging animal viruses.     

Human Monoclonal Antibodies against Highly Conserved HR1 and HR2 Domains of the SARS-CoV Spike Protein Are More Broadly Neutralizing

Immune sera from convalescent patients have been shown to be effective in the treatment of patients infected with Severe Acute Respiratory Syndrome Virus (SARS-CoV) making passive immune therapy with human monoclonal antibodies an attractive treatment strategy for SARS. Previously, using Xenomouse (Amgen British Columbia Inc), we produced a panel of neutralizing Human monoclonal antibodies (HmAbs) that could specifically bind to the ectodomain of the SARS-CoV spike (S) glycoprotein. Some of the HmAbs were S1 domain specific, while some were not. In this study, we describe non-S1 binding neutralizing HmAbs that can specifically bind to the conserved S2 domain of the S protein. However, unlike the S1 specific HmAbs, the S2 specific HmAbs can neutralize pseudotyped viruses expressing different S proteins containing receptor binding domain sequences of various clinical isolates. These data indicate that HmAbs which bind to conserved regions of the S protein are more suitable for conferring protection against a wide range of SARS-CoV variants and have implications for generating therapeutic antibodies or subunit vaccines against other enveloped viruses.     

A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an important emerging virus that is highly pathogenic in aged populations and is maintained with great diversity in zoonotic reservoirs. While a variety of vaccine platforms have shown efficacy in young-animal models and against homologous viral strains, vaccine efficacy has not been thoroughly evaluated using highly pathogenic variants that replicate the acute end stage lung disease phenotypes seen during the human epidemic. Using an adjuvanted and an unadjuvanted double-inactivated SARS-CoV (DIV) vaccine, we demonstrate an eosinophilic immunopathology in aged mice comparable to that seen in mice immunized with the SARS nucleocapsid protein, and poor protection against a nonlethal heterologous challenge. In young and 1-year-old animals, we demonstrate that adjuvanted DIV vaccine provides protection against lethal disease in young animals following homologous and heterologous challenge, although enhanced immune pathology and eosinophilia are evident following heterologous challenge. In the absence of alum, DIV vaccine performed poorly in young animals challenged with lethal homologous or heterologous strains. In contrast, DIV vaccines (both adjuvanted and unadjuvanted) performed poorly in aged-animal models. Importantly, aged animals displayed increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication. These data raise significant concerns regarding DIV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing DIV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.     

Molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms

Massive numbers of palm civets were culled to remove sources for the reemergence of severe acute respiratory syndrome (SARS) in Guangdong Province, China, in January 2004, following SARS coronavirus detection in market animals. The virus was identified in all 91 palm civets and 15 raccoon dogs of animal market origin sampled prior to culling, but not in 1,107 palm civets later sampled at 25 farms, spread over 12 provinces, which were claimed to be the source of traded animals. Twenty-seven novel signature variation residues (SNVs) were identified on the spike gene and were analyzed for their phylogenetic relationships, based on 17 sequences obtained from animals in our study and from other published studies. Analysis indicated that the virus in palm civets at the live-animal market had evolved to infect humans. The evolutionary starting point was a prototype group consisting of three viral sequences of animal origin. Initially, seven SNV sites caused six amino acid changes, at positions 147, 228, 240, 479, 821, and 1080 of the spike protein, to generate low-pathogenicity viruses. One of these was linked to the first SARS patient in the 2003-2004 period. A further 14 SNVs caused 11 amino acid residue changes, at positions 360, 462, 472, 480, 487, 609, 613, 665, 743, 765, and 1163. The resulting high-pathogenicity groups were responsible for infections during the so-called early-phase epidemic of 2003. Finally, the remaining six SNVs caused four amino acid changes, at positions 227, 244, 344, and 778, which resulted in the group of viruses responsible for the global epidemic.     

Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.     

Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain NL63

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.     

Bats and Viruses： a Brief Review

Bats, probably the most abundant, diverse and geographically dispersed vertebrates on earth, have recently been shown to be the reservoir hosts of a number of emerging viruses responsible for severe human and livestock disease outbreaks. Flying foxes have been demonstrated to be the natural reservoir for Hendra and Nipah viruses. Evidence supporting the possibility of bats as potential reservoirs for SARS coronavirus (SARS-CoV) and Ebola virus has also been reported. The recent discovery of these viruses and other viruses occurring naturally in the bat population provides a unique insight into a diverse pool of potentially emergent and pathogenic viruses. The factors which influence the ability of zoonotic viruses to effectively cross the species barrier from bats to other animal populations are poorly understood. A brief review is provided here on the recently emerged bat viruses and on current and future strategies for research in this area.     

Pathogenicity and transmissibility of 2019-nCoV—A quick overview and comparison with other emerging viruses

A zoonotic coronavirus, tentatively labeled as 2019-nCoV by the World Health Organization (WHO), has been identified as the causative agent of the viral pneumonia outbreak in Wuhan, China, at the end of 2019. Although 2019-nCoV can cause a severe respiratory illness like SARS and MERS, evidence from clinics suggested that 2019-nCoV is generally less pathogenic than SARS-CoV, and much less than MERS-CoV. The transmissibility of 2019-nCoV is still debated and needs to be further assessed. To avoid the 2019-nCoV outbreak turning into an epidemic or even a pandemic and to minimize the mortality rate, China activated emergency response procedures, but much remains to be learned about the features of the virus to refine the risk assessment and response. Here, the current knowledge in 2019-nCoV pathogenicity and transmissibility is summarized in comparison with several commonly known emerging viruses, and information urgently needed for a better control of the disease is highlighted.     

Evaluation of affymetrix severe acute respiratory syndrome resequencing GeneChips in characterization of the genomes of two strains of coronavirus infecting humans

Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses.     

Evaluation of Affymetrix Severe Acute Respiratory Syndrome Resequencing GeneChips in Characterization of the Genomes of Two Strains of Coronavirus Infecting Humans

Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses.     

Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease

SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease.     

Increased envelope spike density and stability are not required for the neutralization resistance of primary human immunodeficiency viruses.

Previous observations that the gp120 envelope glycoprotein contents of some primary, clade B human immunodeficiency virus type 1 (HIV-1) isolates were higher than those of laboratory-passaged HIV-1 isolates suggested the hypothesis that increased envelope glycoprotein spike density or stability contributes to the relative neutralization resistance of the primary viruses. To test this, the structural, replicative, and neutralization properties of a panel of recombinant viruses with HIV-1 envelope glycoproteins from divergent clades were examined in an env complementation assay. In this system, although the spike density and stability of envelope glycoproteins from primary HIV-1 isolates were not greater than those from a laboratory-adapted isolate, relative resistance to neutralizing antibodies and soluble CD4 was observed for the viruses with primary envelope glycoproteins. Thus, neither high envelope glycoprotein spike density nor stability is necessary for the relative neutralization resistance of primary HIV-1 viruses.     

ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.     

Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway

Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.