Altering an Artificial Gagpolnef Polyprotein and Mode of ENV Co-Administration Affects the Immunogenicity of a Clade C HIV DNA Vaccine

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ+ CD8+ T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2d T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials

Analysen zur Funktion des E1B-55K-Proteins von Adenovirus Typ 5 im lytischen Replikationszyklus

Das E1B-55K-Protein von Adenovirus Typ 5 ist ein multifunktionelles Phosphoprotein, das eine zentrale Rolle im produktiven Replikationszyklus von Ad5 einnimmt. Die lytischen Aktivitäten des E1B-Proteins werden zumindest teilweise im Komplex mit dem viralen Protein E4orf6 vermittelt. Soweit bekannt fördert der E1B-55K/E4orf6-Komplex (E1B/E4-Komplex) den nukleozytoplasmatischen Transport viraler mRNAs und fördert somit die Synthese viraler Kapsidproteine bzw. die Produktion von Nachkommenviren. Darüber hinaus werden dem E1B/E4-Komplex auch posttranslationale Funktionen beim proteasomalen Abbau des zellulären Tumorsuppressorproteins p53 und dem zellulären DNA-Reparaturkomplex MRN (Mre11, Rad50, NBS1) zugeordnet. Es gibt erste Hinweise, dass beide Aktivitäten mit einer nukleozytoplasmatischen Pendelfunktion in Verbindung stehen, die über ein CRM1-abhängiges Leucin-reiches Kernexportsignal (NES) in beiden viralen Proteinen und ein zum E1B-NES eng benachbartes SUMO1-Konjugationsmotiv (SKM) reguliert wird. Die vorliegende Arbeit befasste sich mit Untersuchungen zur Funktion der E1B-55K- und E4orf6-Proteine in der Regulation des produktiven Replikationszyklus von Ad5, und insbesondere mit Fragestellungen zur Regulation des viralen mRNA-Transports durch den E1B/E4-Komplex. Dazu wurden im ersten Schritt, mit Hilfe eines direkten Klonierungsverfahrens, Mutationen in die kodierenden Bereiche der E1B- und/oder E4orf6-Gene im Ad5-Genom eingeführt, die zu Aminosäureaustauschen in: (1) den NESs von E1B-55K und/oder E4orf6, (2) im SKM von E1B-55K und (3) in den p53- bzw. MRN-Interaktionsdomänen von E1B-55K führen. Anschließend wurde der Einfluss der Mutationen auf die virale Replikation überprüft. Insgesamt bestätigen diese Versuche die Annahme, dass E1B-55K und vermutlich der E1B-55K/E4orf6-Komplex in lytisch infizierten Zellen kontinuierlich über den Exportrezeptor CRM1 aus dem Zellkern transportiert wird. Im Unterschied zu E4orf6 führen Mutationen im E1B-NES zu einer fast vollständigen Restriktion des viralen Proteins im Zellkern, wo das virale Protein an der Peripherie der sog. viralen Replikationszentren akkumuliert. Interessanterweise wird die nukleäre Restriktion und Lokalisation an den viralen Replikationszentren durch die gleichzeitige Mutation des SKM vollständig aufgehoben. Diese Beobachtungen zeigen erstmals, dass E1B-55K posttranslational durch SUMOylierung an die Orte der viralen DNA-Synthese, Transkription und RNA-Prozessierung dirigiert wird und lassen zudem vermuten, dass die Konjugation mit SUMO1 einen CRM1-unabhängigen Exportweg des viralen Proteins reguliert. Trotz der deutlich veränderten Lokalisation des E1B-55K-Proteins haben Mutationen im E4orf6- und/oder E1B-NES sowie im E1B-SKM keinen negativen Einfluss auf die zytoplasmatische Akkumulation viraler Transkripte, Synthese später Strukturproteine, Produktion von Nachkommenviren und den proteasomalen Abbau von MRN. Dieser Befund ist überraschend und widerspricht vollständig der momentan vorherrschenden Modellvorstellung. Obwohl die molekulare Grundlage dieser Ergebnisse noch unklar ist, zeigen die Untersuchungen dieser Arbeit, dass der nukleäre Export von E1B-55K und E4orf6 auch über die CRM1-unabhängigen RNA-Exportrezeptoren HuR und/oder TAP/NXF1 gesteuert wird, da sowohl die HuR-Liganden pp32 und APRIL als auch das TAP/NXF1-Adaptorprotein Aly/REF1 an den E1B/E4-Komplex binden. Während pp32 bekanntermaßen über E4orf6 mit dem E1B/E4-Komplex wechselwirkt, zeigen Koimmunpräzipitationsversuche erstmalig, dass APRIL und Aly/REF1 über E1B-55K mit dem viralen Proteinkomplex interagieren. Eine direkte Beteiligung von Aly/REF1 am RNA-Exportvorgang wird durch Immunfluoreszenzanalysen unterstützt, die zeigen, dass das RNA-bindende hnRN-Protein an die Peripherie der viralen Replikationszentren rekrutiert wird. Außerdem wurden Hinweise erhalten, dass sowohl p53 als auch MRN an diesen Vorgängen direkt oder indirekt beteiligt sind, da der Verlust des proteasomalen Abbaus dieser zellulären Faktoren über den E1B/E4-Komplex mit einer Reduktion später viraler mRNAs und einer stark verringerten Synthese später viraler Proteine sowie Produktion von Nachkommenviren korreliert. Weiterhin wurden im Verlauf dieser Arbeit erstmals zwei neue Isoformen des E1B-55K-Proteins (E1B-48K und E1B-49K) identifiziert. Nach einem vorläufigen Modell werden diese aminoterminal verkürzten E1B-Proteine aufgrund einer sog. internal ribosomal entry site (IRES) und/oder durch den Vorgang des leaky scanning während der Translation der E1B-55K-mRNA gebildet. Die Phänotypisierung einer E1B-55K-negativen Virusmutante, die nur E1B-48K und E1B-49K bildet, zeigt, dass die Expression beider Isoformen für die effiziente virale Replikation ausreichen

A 49-Kilodalton Isoform of the Adenovirus Type 5 Early Region 1B 55-Kilodalton Protein Is Sufficient To Support Virus Replication▿

The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48- to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins (E1B-433R) are sufficient for a productive infection

Influence of the modified constructs on Gag- and Pol-specific CD8⁺ T cell immune responses.

<p>BALB/c mice (n = 6 per group) were i.m. inoculated with equimolar amounts of the indicated plasmid DNA constructs (80 µg of ^ΔMGPN, ^MGPN, ^ΔMG^FSPN, ^MG^FSPN; 57 µg of ^MGag; 68 µg of PN). After 12 days, spleen cells were isolated and tested for specific cellular immune responses by measuring IFNγ production after stimulation with Gag (black) or Pol (grey) specific peptides. IFNγ production was determined by FACS analysis after intracellular staining of IFNγ. Cell culture medium served as a negative control. (A) Influence of budding competence and the functional frameshift on Gag- and Pol-specific CD8⁺ T cell immune responses. (B) Impact of separating the Gag and PolNef reading frames on Gag- and Pol-specific CD8⁺ T cell responses. Data shown are representative of two experiments.</p

Spatially separating Gag-Pol-Nef and gp120 alters Gag- and Pol-specific CD8⁺ T cell responses.

<p>BALB/c mice (n = 6 per group) were i.m. inoculated (+) in both legs with an equimolar mixture of ^MGag and PN; gp120 alone, or a mixture of ^MGag, PN and gp120, where the amount of gp120 was titrated (mixture: 57, 30, 10 µg). Further groups received plasmid DNAs spatially separated as a mixture of ^MGag and PN in the right leg and titrated amounts (separate: 57, 30, 10 µg) of gp120 in the left leg. After 12 days, spleen cells were isolated and tested for specific cellular immune responses by measuring IFNγ production after stimulation with Gag (black), Pol (grey) or Env (hatched) specific peptides. IFNγ production was determined by FACS analysis after intracellular staining of IFNγ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034723#pone-0034723-g003" target="_blank">Fig. 3</a>. Cell culture medium served as a negative control. Data shown are representative of two experiments.</p

Expression of Gag and Env in co-transfected 293T cells and the effect of administrating Gag-2a-Env on Gag- and Env-specific CD8⁺ T cell responses.

<p>(A) 293T cells were transiently transfected with 3 µg of the indicated plasmid DNA constructs. Following 48 h incubation, TCA-precipitated supernatants and cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using p24-specific (Gag) mouse mAb (CB-4/1), and gp120-specific mouse mAb (MH23), as indicated. Molecular weight markers and positions of the detected proteins are indicated. mock: non-transfected 293T cells. (B) 293T cells were transiently transfected with equimolar amounts of the indicated plasmid DNA constructs. At 48 h post transfection, cells were permeabilized, stained with anti-p24-PE and/or anti-gp120-ALEXA 488 and analyzed by flow cytometry. (C) BALB/c mice (n = 6 per group) were inoculated i.m. with (i) an equimolar mixture of ^MGag and gp120 in both legs, (ii) ^MGag in the left, and gp120 in the right leg, or (iii) ^MGag-2a-gp120. After 12 days, spleen cells were isolated and tested for specific cellular immune responses by measuring IFNγ production after stimulation with Gag (black) and Env (hatched) specific peptides as above. IFNγ production was determined using FACS analysis after intracellular staining of IFNγ. Cell culture medium served as negative control. Data shown are representative of two experiments.</p

Protein expression from the modified constructs.

<p>293T cells were transiently transfected with the indicated plasmid DNA constructs using jetPEI. Cells and supernatants were harvested after 48 h. Cell lysates, and supernatants processed by TCA/acetone precipitation, were separated by 8.0% SDS-PAGE and analyzed by immunoblotting using p24-specific (Gag) mouse mAb (CB-4/1), HIV-1 RT (Pol)-specific mouse mAb (5B2B2), and gp120-specific mouse mAb (MH23) to specifically detect the corresponding recombinant polypeptides. Molecular weight markers and positions of the specifically detected proteins are indicated. mock: non-transfected 293T cells.</p

Impact of deleting the BALB/c immunodominant gp120 epitope V11V on Gag-specific CD8⁺ T cell immune responses.

<p>BALB/c mice (n = 6 per group) were i.m. inoculated with (i) equimolar amounts of the indicated plasmid DNA construct (57 µg of ^MGag, gp120, gp120^ΔV11V), (ii) an equimolar mixture of ^MGag and gp120, or (iii) an equimolar mixture of ^MGag and gp120^ΔV11V. After 12 days, spleen cells were isolated and tested for specific cellular immune responses by measuring IFNγ production after stimulation with Gag (black) or Env (hatched) specific peptides. IFNγ production was determined using FACS analysis after intracellular staining of IFNγ. Cell culture medium served as negative control. Data shown are representative of two experiments.</p

Effect of co-immunization with Gag-Pol-Nef and gp120 on Gag- and Pol-specific CD8⁺ T cell responses.

<p>BALB/c mice (n = 6 per group) were i.m. inoculated (+) with ^MG^FSPN; or equimolar mixtures of ^MG^FSPN and gp120; ^MGag and PN; or ^MGag, PN, and gp120. After 12 days, spleen cells were isolated and tested for specific cellular immune responses by measuring IFNγ production after stimulation with Gag (black), Pol (grey) or Env (hatched) specific peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034723#pone-0034723-g003" target="_blank">Fig. 3</a>. Cell culture medium served as a negative control. Data shown are representative of two experiments.</p