Non-redundant and Redundant Roles of Cytomegalovirus gH/gL Complexes in Host Organ Entry and Intra-tissue Spread

Author Summary The role of viral glycoprotein entry complexes in viral tropism in vivo is a question central to understanding virus pathogenesis and transmission for any virus. Studies were limited by the difficulty in distinguishing between viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. Employing the murine cytomegalovirus entry complex gH/gL/gO as a paradigm for a generally applicable strategy to dissect these two events experimentally, we used a gO-transcomplemented ΔgO mutant for providing the complex exclusively for the initial cell entry step. In immunocompromised mice as a model for recipients of hematopoietic cell transplantation, our studies revealed an irreplaceable role for gH/gL/gO in initiating infection in host organs relevant to pathogenesis, whereas subsequent spread within tissues and infection of the salivary glands, the site relevant to virus host-to-host transmission, are double-secured by the entry complexes gH/gL/gO and gH/gL/MCK-2. As an important consequence, interventional strategies targeting only gO might be efficient in preventing organ manifestations after a primary viremia, whereas both gH/gL complexes need to be targeted for preventing intra-tissue spread of virus reactivated from latency within tissues as well as for preventing the salivary gland route of host-to-host transmission

Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper

The principal focus of this work was the dissection of the humoral immune response against the murine cytomegalovirus (mCMV). mCMV was chosen, because it can be considered a model system for the infection and pathogenesis of the human cytomegalovirus (hCMV). An infection with hCMV is currently the most frequent vertically transmitted infectious disease. Many newborns suffer from long-term medical conditions caused by hCMV infection. hCMV infection is also a leading cause of serious complications, morbidity and mortality in immunosuppressed transplant recipients or AIDS patients. The analysis of the humoral immune response directed against viral glycoproteins, above all against glycoprotein B (gB), was of particular interest. The problem was addressed with two different approaches. Firstly, monoclonal antibodies were generated from B-cells of infected mice using the hybridoma technique. These antibodies were then analyzed for their antiviral potential in vitro, that is, their capability to inhibit infection of susceptible target cells in cell culture. Many isolated antibodies were able to neutralize mCMV in vitro. Most of the antibodies, however, were non-neutralizing. We further successfully identified the target structures of antiviral antibodies. The majority of neutralizing antibodies was directed against antigenic domains on gB. One antiviral antibody that was extraordinarily potent in vitro recognized yet another envelope glycoprotein, gN. The 3D structure of gB had been modeled computationally. A structural domain shown therein was demonstrated to comprise the epitope of a virus neutralizing antibody, which was isolated in this work. In addition, the epitope of another antiviral antibody was identified. Its homologous domain in hCMV (AD-1) has been described to be highly immunogenic. When determining the seropositivity rate against the two domains, the AD-1 homologous domain was demonstrated to be recognized by 100% of mCMV infected animals. DomII was also highly immunogenic. 90% of all infected mice mounted antibody responses against DomII. These findings correspond to the immune response of hCMV infected individuals against these domains. In vivo experiments focused on one DomII-specific antibody with neutralizing activity (1F11) and one non-neutralizing AD-1-specific antibody (5F12). Additionally the in vivo antiviral activity of the gN-specific antibody (32.22) was analyzed. All three antibodies were found to transfer protection against lethal challenge with mCMV when present at the time of infection. It is even possible, that sterilizing immunity was provided. Moreover, the neutralizing antibodies achieved a reduction of the viral load in mice with an already established infection. Thus, in this study for first time mCMV-specific antibodies with known protein targets were isolated that are able to protect against infection with mCMV. The second approach focused on the immunization with the described immunogenic domains of gB. For this, DomII and AD-1 were prokaryotically expressed as GST fusion proteins and purified. Mice were immunized in a prime-boost fashion and the immune response towards these antigens was monitored. Both proteins induced specific antibodies following vaccination. DomII was shown to be particularly immunogenic. The induced antibodies, however, were not able to prevent the infection of susceptible target cells in vitro. Also, the transfer of DomII-specific B-cells or a specific serum pool did not provide protection against infection in vivo. The immunization with UV-inactivated mCMV, on the other hand, led to the induction of antibodies with antiviral activity. This shows that protective immunity against mCMV infection can be induced. It will be the concern of future studies to modify the candidate vaccines in a way that they prompt protective antibody titers.In dieser Arbeit wurde die humorale Immunantwort im Schutz gegen die Infektion mit dem murinen Cytomegalievirus (mCMV) untersucht. mCMV diente hierbei als Modellsystem für die Infektion und Pathogenese des humanen Cytomegalievirus (hCMV). Die Infektion mit hCMV ist heute die häufigste vertikal übertragene Infektionskrankheit und viele Neugeborene leiden unter Langzeitbeschwerden, die durch die Infektion hervorgerufen werden können. Zudem kann der Ausbruch von hCMV in Empfängern von Organtransplantaten oder AIDS Patienten zu schweren Komplikationen führen. Von besonderem Interesse für diese Arbeit war die Analyse der Immunantwort gegen virale Glykoproteine, in erster Linie gegen das immundominante Glykoprotein B (gB). Dafür wurden zwei Arbeitsansätze ausgewählt. Im ersten Arbeitsansatz wurden mittels Hybridomtechnik monoklonale Antikörper aus B-Zellen infizierter Mäuse erfolgreich hergestellt. Diese Antikörper wurden in vitro auf ihr Potential hin untersucht mCMV zu neutralisieren, das heißt die Infektion suszeptibler Zielzellen zu verhindern. Viele isolierte Antikörper zeigten diese Fähigkeit, jedoch waren die meisten Antikörper nicht neutralisierend. Zudem wurden die Zielproteine antiviraler Antikörper identifiziert und die Positionen der Epitope darauf genauer eingegrenzt. Es stellte sich heraus, dass der Großteil der neutralisierenden Antikörper, die während einer mCMV Infektion hergestellt werden, gegen gB gerichtet ist. Ein weiterer Antikörper, der in vitro besonders potente antivirale Eigenschaften zeigte, erkannte Glykoprotein N (gN), das wie gB in der Virushülle eingelagert ist. Mit Hilfe bioinformatischer Methoden wurde die 3D Struktur von gB modelliert. Eine hierbei identifizierte strukturelle Domäne (DomII) enthält das Epitop eines in dieser Arbeit beschriebenen neutralisierenden Antikörpers. Des Weiteren wurde in Homologie zu hCMV der C-terminale Bereich AD-1 als Bindestelle für einen antiviralen Antikörper identifiziert. Die Analyse von Seren infizierter Tiere zeigte eine Seropositivitätsrate von 100% für die Erkennung von AD-1. DomII wurde von 90% der Seren erkannt. Dies entspricht der Seropositivitätsrate von infizierten Personen gegen die entsprechenden Domänen auf hCMV. Zwei in vitro neutralisierende Antikörper und ein nicht—neutralisierender Antikörper wurden ausgewählt und für in vivo Versuche erfolgreich aufgereinigt. Der nicht-neutralisierende Antikörper erkannte AD-1. Ein neutralisierender Antikörper war gegen DomII gerichtet und der andere erkannte ein Epitop auf gN. In Schutzversuchen zeigte sich, dass alle drei getesteten Antikörper die Infektion mit mCMV verhindern können, wenn sie zum Zeitpunkt der Infektion vorhanden sind. Wahrscheinlich wurde hier sogar sterilisierende Immunität erzielt. In der Therapie einer etablierten mCMV Infektion konnten die neutralisierenden Antikörper die Infektion eindämmen und einen Überlebensvorteil bewirken. In dieser Arbeit wurden somit zum ersten Mal mCMV spezifische Antikörper mit in vivo antiviralen Eigenschaften und bekannter Epitopspezifität beschrieben. Der zweite Arbeitsansatz befasste sich mit Immunisierungsstudien, basierend auf Fragmenten von gB. Dafür wurden die relevanten Bereiche (AD-1 und DomII) im prokaryonten System als Fusionsproteine aufgereinigt und in ‚Prime-Boost‘ Schritten an die Tiere verabreicht. Beide Proteine induzierten nach der Immunisierung spezifische Antikörper. DomII stellte sich dabei als außergewöhnlich immunogen heraus. Die induzierten DomII oder AD-1 spezifischen Antikörper konnten jedoch im in vitro System die Infektion suszeptibler Zellen nicht verhindern. Die Verabreichung von DomII-spezifischen B-Zellen oder eines spezifischen Serumpools zeigte auch in vivo keinen antiviralen Effekt. Dagegen wurden nach Immunisierung mit UV-inaktiviertem mCMV Antikörper induziert, die in der Lage waren die Infektion einzudämmen. Eine sterile Immunität wurde hier jedoch nicht beobachtet. Es bleibt die Aufgabe nachfolgender Studien, die Impfstoffe derart zu modifizieren, dass antivirale Antikörper induziert werden und ein Schutz gegen die Infektion mit mCMV erreicht wird

Non-redundant and Redundant Roles of Cytomegalovirus gH/gL Complexes in Host Organ Entry and Intra-tissue Spread

Abstract Herpesviruses form different gH/gL virion envelope glycoprotein complexes that serve as entry complexes for mediating viral cell-type tropism in vitro; their roles in vivo, however, remained speculative and can be addressed experimentally only in animal models. For murine cytomegalovirus two alternative gH/gL complexes, gH/gL/gO and gH/gL/MCK-2, have been identified. A limitation of studies on viral tropism in vivo has been the difficulty in distinguishing between infection initiation by viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. As a new strategy to dissect these two events, we used a gO-transcomplemented ΔgO mutant for providing the gH/gL/gO complex selectively for the initial entry step, while progeny virions lack gO in subsequent rounds of infection. Whereas gH/gL/gO proved to be critical for establishing infection by efficient entry into diverse cell types, including liver macrophages, endothelial cells, and hepatocytes, it was dispensable for intra-tissue spread. Notably, the salivary glands, the source of virus for host-to-host transmission, represent an exception in that entry into virus-producing cells did not strictly depend on either the gH/gL/gO or the gH/gL/MCK-2 complex. Only if both complexes were absent in gO and MCK-2 double-knockout virus, in vivo infection was abolished at all sites. Author Summary The role of viral glycoprotein entry complexes in viral tropism in vivo is a question central to understanding virus pathogenesis and transmission for any virus. Studies were limited by the difficulty in distinguishing between viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. Employing the murine cytomegalovirus entry complex gH/gL/gO as a paradigm for a generally applicable strategy to dissect these two events experimentally, we used a gO-transcomplemented ΔgO mutant for providing the complex exclusively for the initial cell entry step. In immunocompromised mice as a model for recipients of hematopoietic cell transplantation, our studies revealed an irreplaceable role for gH/gL/gO in initiating infection in host organs relevant to pathogenesis, whereas subsequent spread within tissues and infection of the salivary glands, the site relevant to virus host-to-host transmission, are double-secured by the entry complexes gH/gL/gO and gH/gL/MCK-2. As an important consequence, interventional strategies targeting only gO might be efficient in preventing organ manifestations after a primary viremia, whereas both gH/gL complexes need to be targeted for preventing intra-tissue spread of virus reactivated from latency within tissues as well as for preventing the salivary gland route of host-to-host transmission

Synchronicity of infection initiation in main cell types of the liver.

<p>(A) 2C-IHC of liver tissue sections taken at 24h after i.v. infection of immunocompromised BALB/c mice (6.5 Gy of γ-irradiation) with 1 x 10⁶ PFU of WT mCMV, simultaneously detecting viral proteins IE1 (black staining) and E1 (red staining) in nuclei of infected cells. Left image: overview. iEC, infected endothelial cell; iHc, infected hepatocyte. The framed area is shown enlarged in the right image. Bar markers represent 25 μm. (B) Cell counts in representative 10-mm² areas of liver tissue sections quantitating IE1⁺E1^- and IE1⁺E1⁺ cells differentiated by cell type as indicated. Infection had not proceeded in any cell type to expression of gB, viral DNA synthesis (vDNA detected by ISH), and the late (L) phase protein MCP. Symbols represent linked data from livers of 3 mice analyzed individually. The median values are marked.</p

Double-ko of gO and MCK-2 ablates <i>in vivo</i> virus growth.

<p>(A) Sketch with WT and ΔgOΔMCK-2-gO^trans virion pictograms explaining the gH/gL complex envelope equipment of viruses upon first cell entry (incoming virions) and of their progeny participating in subsequent intra-tissue spread. (B) Time course of counts of infected IE1⁺ liver cells (outer left panel) or of qPCR-determined viral genome loads in liver, spleen, and lungs (remaining panels) after i.v. infection of immunocompromised BALB/c mice (6.5 Gy of γ-irradiation) with 10³ PFU of WT virus (filled circles) or ΔgOΔMCK-2-gO^trans virus (open circles). For the explanation of log-linear regression analysis (calculating vDT), see the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004640#ppat.1004640.g004" target="_blank">Fig. 4</a>.</p

gO-independence of virus spread in liver tissue.

<p>(A) Sketch of the concept with WT and ΔgO (Δm74) virion pictograms explaining the gH/gL complex envelope equipment of viruses upon first cell entry (incoming virions) and of their progeny participating in subsequent intra-tissue spread. (B) Time course of counts of infected IE1⁺ liver cells, all cells or differentiated by cell type, after i.v. infection of immunocompromised BALB/c mice (6.5 Gy of γ-irradiation) with 10³ PFU of WT virus (filled circles) or ΔgO virus (open squares). Symbols represent the median values of cell counts per representative 10-mm² areas of liver tissue sections from at least 3 mice per group and time of assay. Log-linear regression lines (based on all data) and their corresponding 95% confidence areas (bordered by dotted lines) are indicated. Viral doubling times (vDT) were calculated based on the slopes <i>a</i> of the regression lines according to the formula vDT = log2/<i>a</i>. The 95% confidence intervals of vDT are given in parentheses. (C) 2C-IHC images taken on day 10 after infection with WT virus (left panels) or ΔgO virus (right panels). Upper two images show representative tissue section areas stained for IE1 (red) and the macrophage marker F4/80 (turquoise green). Lower two images show representative tissue section areas stained for IE1 (red) and the EC marker CD31 (black). The bar marker represents 100 μm and applies to all 4 images.</p

Redundance of alternative gH/gL complexes gH/gL/gO and gH/gL/MCK-2 in securing the infection of salivary glands.

<p>(A) Time course of SG infection (for conditions and qPCR assay see the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004640#ppat.1004640.g005" target="_blank">Fig. 5</a>) by viruses WT (filled circles), ΔgO (open squares), and ΔgOΔMCK-2-gO^trans (open circles). (B) Independent second experiment reproducing the time course of SG infection by viruses WT (filled circles) and ΔgO (open squares), now compared to virus ΔMCK-2 (open circles) still expressing the gH/gL/gO complex. Symbols in the three single virus panels represent data from individual mice, symbols in the merge (outer right) panel represent the corresponding median values. For the explanation of log-linear regression analysis (calculating vDT), see the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004640#ppat.1004640.g004" target="_blank">Fig. 4</a>.</p

Requirement of gO for efficient initiation of infection in diverse liver cell types.

<p>(A) Sketch of liver tissue microanatomy with the localization of Hc, EC (black stain), and MΦ (turquoise-green stain). Infection of cells is symbolized by viral IE1 protein-containing cell nuclei (red stain). (B) 3C-IHC images of liver tissue sections taken at 24h after i.v. infection of immunocompromised BALB/c mice (6.5 Gy of γ-irradiation) with 1 x 10⁶ PFU of WT mCMV. (a) Overview showing infected Hc (iHc, red stained nucleus, IE1 protein), infected IE1⁺ (red) F4/80⁺ (turquoise green) MΦ (iMΦ), and infected IE1⁺ (red) CD31⁺ (black) EC (iEC). (b) Higher magnification image showing iMΦ and iHc in greater detail. (c) Higher magnification image showing iEC and a binucleated iHc in greater detail. Bar markers: 25 μm. (C) Counts of infected IE1⁺ cells (sum of IE1⁺E1^- and IE1⁺E1⁺ cells) of the indicated liver cell types in representative 10-mm² areas of liver tissue sections after infection with viruses WT or ΔgO (Δm74) under the conditions specified above. Symbols represent data (linked data within each infection group) from individual mice with the median values marked.</p

Reversal of the ΔgO growth deficiency phenotype by gO-transcomplementation.

<p>(A) Sketch with WT and ΔgO-gO^trans virion pictograms explaining the gH/gL complex envelope equipment of viruses upon first cell entry (incoming virions) and of their progeny participating in subsequent intra-tissue spread. (B) Time course of counts of infected IE1⁺ liver cells, all cells or differentiated by cell type, after i.v. infection of immunocompromised BALB/c mice (6.5 Gy of γ-irradiation) with 10³ PFU of WT virus (filled circles) or virus ΔgO-gO^trans (filled squares). Symbols represent the median values of cell counts per representative 10-mm² areas of liver tissue sections from at least 3 mice per group and time of assay. (C) Corresponding analysis of viral DNA load in spleen and lungs (mean of triplicate tissue samples per mouse) by qPCR specific for gene M55 (encoding gB), with qPCR specific for cellular gene <i>pthrp</i> performed for normalization to host cell numbers. Symbols represent median values from at least 3 individually tested mice per group and time of assay. For the explanation of log-linear regression analysis (calculating vDT), see the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004640#ppat.1004640.g004" target="_blank">Fig. 4</a>.</p