Natural selection supports escape from concerted evolution of a recently duplicated CEACAM1 paralog in the ruminant CEA gene family

Concerted evolution is often observed in multigene families such as the CEA gene family. As a result, sequence similarity of paralogous genes is significantly higher than expected from their evolutionary distance. Gene conversion, a "copy paste" DNA repair mechanism that transfers sequences from one gene to another and homologous recombination are drivers of concerted evolution. Nevertheless, some gene family members escape concerted evolution and acquire sufficient sequence differences that orthologous genes can be assigned in descendant species. Reasons why some gene family members can escape while others are captured by concerted evolution are poorly understood. By analyzing the entire CEA gene family in cattle (Bos taurus) we identified a member (CEACAM32) that was created by gene duplication and cooption of a unique transmembrane domain exon in the most recent ancestor of ruminants. CEACAM32 shows a unique, testis-specific expression pattern. Phylogenetic analysis indicated that CEACAM32 is not involved in concerted evolution of CEACAM1 paralogs in ruminants. However, analysis of gene conversion events revealed that CEACAM32 is subject to gene conversion but remarkably, these events are found in the leader exon and intron sequences but not in exons coding for the Ig-like domains. These findings suggest that natural selection hinders gene conversion affecting protein sequences of the mature protein and thereby support escape of CEACAM32 from concerted evolution

EVALUATION OF EFFICACY OF NIBRG-14 VACCINE AGAINST HIGHLY PATHOGENIC H5N1 VIRUSES ISOLATED DURING 2011 INFLUENZA OUTBREAKS IN VIETNAM

Highly pathogenic avian  influenza (HPAI) H5N1 viruses continue to be endemic  in many Asian countries causing  lethal  infections  in human. The vaccine virus  (NIBRG-14) developed from a H5N1 virus strain (A/Vietnam/1194/2004) has been approved by WHO for use in human as  well  as  poultry  vaccine.  It  is  well-known  that  the  A/H5N1  viruses  have  diversified  both genetically  and  antigenically  allowing  them  to  escape  from  the  host  immune  surveillance system.  Therefore,  evaluation  of  the  vaccine  immunogenicity  and  its  relationship  to  newly emerging  viruses  is  crucially  important. NIBRG-14 virus particles propagated  in  embryonated chicken eggs were inactivated with formalin and adjuvanted with mineral oil to form a water-in-oil emulsion. The  resulting vaccine was  injected  subcutaneously  into chickens and ducks. The vaccinated birds were challenged with  the HPAI virus strains circulating  in Vietnam  including clade  1,  clade  2.3.2.1a  and  2.3.2.1b  at  day  21  post-vaccination  (p.  v.).  We  observed  that vaccinated birds were protected from manifestation of disease signs upon challenge with HPAI clade 1 and clade 2.3.2.1a viruses; however,  it did not confer protection against clade 2.3.2.1b challenge  andstressing  the  need  for  development  of  new  effective  vaccines  against  the  newly emerging viruses

Immunization with GP1 but Not Core-like Particles Displaying Isolated Receptor-Binding Epitopes Elicits Virus-Neutralizing Antibodies against Junín Virus

New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses.Fil: Roman Sosa, Gleyder. Ulm University Hospital; AlemaniaFil: Leske, Anne. Friedrich-Loeffler-Institut; AlemaniaFil: Ficht, Xenia. Ulm University Hospital; AlemaniaFil: Dau, Tung Huy. Friedrich-Loeffler-Institut; AlemaniaFil: Holzerland, Julia. Friedrich-Loeffler-Institut; AlemaniaFil: Hoenen, Thomas. Friedrich-Loeffler-Institut; AlemaniaFil: Beer, Martin. Friedrich-Loeffler-Institut; AlemaniaFil: Kammerer, Robert. Friedrich-Loeffler-Institut; AlemaniaFil: Schirmbeck, Reinhold. Friedrich-Loeffler-Institut; AlemaniaFil: Rey, Felix A.. Friedrich-Loeffler-Institut; AlemaniaFil: Cordo, Sandra Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Groseth, Allison. Friedrich-Loeffler-Institut; Alemani

The role of cell-penetrating peptides in the induction of T cell responses by virus-like particles

Many viral structural proteins can self-assemble into virus-like particles (VLPs). VLPs can serve as an effective vaccine or be used as a vaccine platform. One of these structural proteins is the hepatitis B virus core antigen (HBcAg), which appears to be suitable as an antigen carrier due to its high immunogenicity. HBcAg has a major immunodominant region (MIR) that is presented on the surface of the VLPs after self-assembly. Foreign antigens can be inserted into this region. Since HBcAg VLPs, unlike the Hepatitis B virus (HBV), do not have an envelope, they are not able to penetrate cell membranes efficiently. As an extracellular antigen, HBcAg VLPs primarily induce a strong humoral immune response. In the present study, we investigated the extent to which HBcAg can be modified to also elicit an enhanced cellular, particularly a cytotoxic, immune response. A cytotoxic CD8+ T cell response is predominantly induced by intracellular antigens. Therefore, our goal was to increase the cell penetration capacity of VLPs. We aimed to achieve this by fusing cell-penetrating peptides (CPPs) to HBcAg. CPPs can spontaneously penetrate cell membranes to enter the cytoplasm of cells. To guarantee that the CCPs were localized to the surface of the VLPs, we fused CPPs to the N-terminus of HBcAg. The CCPs were followed by a tag to allow the purification of VLPs. The T cell epitopes, against which the induced CTL should be directed, were derived from the Large T antigen and inserted into the MIR of HBcAg. Finally, we fused fluorescent proteins to the C-terminus of HBcAg to track the entry of VLPs into cells. Modifications of HBcAg may lead to reduced stability or altered structure of VLPs. To analyze the stability of VLPs, we used nanoscale differential scanning fluorimetry (nanoDSF) analysis. This revealed that the N-terminal fusion of CPPs or the tag to HBcAg does not reduce VLP stability. However, some peptides incorporated into the MIR had a significant effect on the structure and stability of the VLPs. While the incorporation of a Flag-tag or a peptide from ovalbumin had no negative effect on VLP stability, the incorporation of peptides representing T cell epitopes of Large T antigen interfered with VLP formation. Denaturation and reassembly of the aggregates significantly improved the homogeneity of the VLPs, and the C-terminal addition of arginine-rich domains enhanced stability. Using live cell imaging and flow cytometry, we demonstrated that HBcAg VLPs functionalized with CPP exhibited up to 40% more efficient penetration into professional antigen-presenting cells (JAWS II) than HBcAg VLPs without CPP. This resulted in the increased presentation of integrated T cell epitopes by dendritic cells. In vivo, we detected significantly increased induction of SV40 Large T antigen-specific CTL in mice immunized with CPP-conjugated VLPs compared to unconjugated VLPs. In this study, we demonstrated that a stronger cellular immune response can be induced by CPP-functionalized HBcAg VLPs than with the unmodified HBcAg VLPs in vitro as well as in vivo. This discovery may have positive implications for future vaccine development where an enhanced cellular component of the immune response is desirable.Viele virale Strukturproteine haben die Fähigkeit sich selbst zu virusähnlichen Partikeln (VLPs) anzuordnen, die als wirksame Impfstoffe dienen oder als Impfstoffplattformen genutzt werden können. Eines dieser Strukturproteine ist das Hepatitis-B-Virus-Core-Antigen (HBcAg), das sich durch seine hohen Immunogenität als Antigenträger-VLPs zu eignen scheint. Das HBcAg verfügt über eine immundominante Region (MIR), die an der Oberfläche der VLPs präsentiert wird. In diese Region können Fremdantigene eingefügt werden. Da das HBcAg VLP im Gegensatz zum Hepatitis B Virus (HBV) keine Hülle besitzt ist es nicht in der Lage effizient in Zellen einzudringen. Als extrazelluläre Antigene führen die HBcAg-VLPs in erster Linie zu einer starken humoralen Immunantwort. In der vorliegenden Studie wurde untersucht in wie weit das HBcAg modifiziert werden kann, um auch eine verstärkte zelluläre, insbesondere einer zytotoxischen, Immunantwort zu erzielen. Eine zytotoxische CD8+ T-Zellantwort wird vorwiegend durch intrazelluläre Antigene hervorgerufen. Das Ziel bestand daher darin die Zellpenetrationskapazität der VLPs zu erhöhen. Dies sollte durch die Fusion von zellpenetrierenden Peptiden (CPPs) an das HBcAg erreicht werden. CPPs haben die Fähigkeit, spontan Zellmembranen zu durchdringen und so ins Zytoplasma von Zellen zu gelangen. Um zu garantieren, dass die CCPs an der Oberfläche der VLPs lokalisiert sind wurde der N-Terminus des HBcAg als Fusionspartner gewählt. Die CCPs wurden von einer Markierung (Tag) gefolgt, die eine Aufreinigung der VLPs ermöglichen sollte. Die T-Zellepitope gegen die die Induzierten CTL gerichtet sein sollten wurden vom SV40 Large T Antigen abgeleitet und in die MIR des HBcAg eingefügt. Schließlich fusionierten wir fluoreszierende Proteine an den C-Terminus des HBcAg um das Eindringen der VLPs in die Zelle einfach verfolgen zu können. Modifikationen des HBcAg können zu einer reduzierten Stabilität oder zu einer veränderten Struktur der VLPs führen, um dies zu kontrollieren wurde die intrinsische Fluoreszenzanalyse mit thermischer Entfaltung verwendet. Dabei hat sich ergeben, dass die N-terminale Fusion von CPPs bzw. des Tags an das HBcAg die VLP-Stabilität nicht reduziert. Überraschender Weise hat sich aber gezeigt, dass die Peptide, die in die MIR eingebaut wurden einen erheblichen Einfluss auf die Struktur und Stabilität der VLPs hatten. Während der Einbau eines Flag-tag oder einem Peptid vom Ovalbumin keinen negativen Auswirkungen auf die VLP Stabilität hatte führte der Einbau der T-Zellepitope des Large T Antigens zu Aggregation der VLPs. Durch Denaturierung und Reassemblierung der Aggregate konnte die Homogenität der VLPs deutlich verbessert werden und durch C-terminales Anfügen Arginin-reicher Domänen konnte die Stabilität positive beeinflusst werden. Mittels Live-Cell-Imaging und durchflußzytometrischen Analysen konnte ich zeigen, dass mit CPP funktionalisierte HBcAg-VLPs ein bis zu 40% effizienteres Eindringen in professionelle Antigenpräsentierenden Zellen (JAWS II) zeigten als HBcAg-VLPs ohne CPP. Dies führte zu einer erhöhten Präsentation der in die MIR integrierten T-Zellepitope durch die dendritischen Zellen. In vivo konnten eine signifikant erhöhte Induktion von Large T Antigen-spezifischen CTL in Mäusen, die mit CPP konjugierten VLPs immunisiert wurden, detektiert werden im Vergleich zu Mäusen, die mit unkonjugierten VLPs immunisiert wurden. In dieser Studie konnte gezeigt werden, dass durch CPP funktionalisierte HBcAg-VLPs in vitro als auch in vivo eine stärkere zelluläre Immunantwort induzieren als nicht modifizierten HBcAg-VLPs. Diese Entdeckung könnte in Zukunft positive Auswirkungen auf die Entwicklung von Vakzinen haben, bei der eine verstärkte zelluläre Komponente der Immunantwort wünschenswert ist