Mosaic chimeric viral vaccine particle
11352416 · 2022-06-07
Assignee
Inventors
- Waithaka Mwangi (College Station, TX, US)
- Surya Waghela (College Station, TX, US)
- Luc Berghman (Collge Station, TX, US)
Cpc classification
C07K16/2851
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C12N2810/855
CHEMISTRY; METALLURGY
C12N2770/24334
CHEMISTRY; METALLURGY
C07K16/1081
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention describes compositions and methods for priming protective immunity in the presence of pre-existing maternal antibody. In some embodiments, the invention contemplates simultaneously masking vaccines to avoid antibody neutralization while targeting those vaccines to specific cell types in order to elicit an enhanced immune response. In other embodiments, vectors that recruit and activate specific antigen-presenting cells may further enhance the efficacy of those immune responses.
Claims
1. A modified live vaccine particle coated with a bi-specific diabody, wherein said bi-specific diabody comprises a virus-masking motif having affinity to at least one of a plurality of antigenic sites on said vaccine particle, said plurality of antigenic sites derived from two or more of the BVDV 1 or BVDV 2 proteins selected from the group consisting of N.sup.pro, capsid, E.sup.ms, E1, E2, NS2 and NS3.
2. The modified live vaccine of claim 1, wherein said at least one of said bi-specific diabody further comprises a cell surface antigen binding moiety fused in-frame with said plurality of antigenic sites.
3. The modified viral vaccine of claim 2, wherein said cell surface antigen binding moiety is a dendritic cell surface antigen binding moiety.
4. The modified live vaccine particle of claim 1, further comprising a single chain antibody having specific affinity for CD205.
5. The modified live vaccine particle of claim 1, wherein said particle is a modified live vectored vaccine.
6. The modified live vaccine particle of claim 1, wherein said bi-specific diabody comprises a CD205-BVDV 1 & 2-specific antibody conjugate.
7. The modified live vaccine of claim 3, wherein said dendritic cell surface antigen binding moiety is a bovine CD205 antigen receptor binding moiety.
8. The modifed live vaccine of claim 2, wherein said viral antigen binding moiety binds to at least one of said plurality of antigenic sites.
9. The modified live vaccine of claim 1, wherein said at least one of said plurality of antigenic sites further comprises a protein fragment derived from a virus selected from the group consisting of bovine respiratory viruses, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, bovine parainfluenza-3 virus, bovine herpes virus 1 and bovine herpes virus 2.
10. A method, comprising: a) providing; i) a subject at risk for infection by a virus, ii) a modified live vaccine particle coated with a bi-specific diabody, wherein said bi-specific antibody comprises a virus-masking motif having affinity to at least one of a plurality of antigenic sites on said vaccine particle, said plurality of antigenic sites derived from two or more of the BVDV 1 or BVDV 2 proteins selected from the group consisting of N.sup.pro, capsid, E.sup.ms, E1, E2, NS2 and NS3; and b) administering said vaccine to said subject under conditions such that protective immunity is induced.
11. The method of claim 10, wherein said modified live vaccine particle further comprises a single chain antibody having specific affinity for CD205.
12. The method of claim 10, wherein said subject is a bovine or a neonatal calf.
13. The method of claim 10, wherein said subject has neutralizing maternal antibodies specific for BVDV-1 or BVDV-2 prior to said administering of said vaccine.
14. The method of claim 13, wherein said vaccine evades said neutralizing maternal antibodies.
15. The method of claim 10, wherein said at least one of said plurality of bi-specific diabodies further comprises a cell surface antigen binding moiety fused in-frame with said viral antigen binding moiety.
16. The method of claim 15, wherein said cell surface antigen binding moiety is a dendritic cell surface antigen binding moiety.
17. The method of claim 16, wherein said dendritic cell surface antigen binds to, activates and recruits a dendritic cell.
18. The method of claim 16, wherein said dendritic cell surface antigen binding moiety is a bovine CD205 antigen receptor binding moiety.
19. The method of claim 15, wherein said viral antigen binding moiety binds to at least one of said plurality of antigenic sites.
20. The method of claim 10, wherein said plurality of antigenic sites further comprises a protein fragment derived from a virus selected from the group consisting of bovine respiratory viruses, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, bovine parainfluenza-3 virus, bovine herpes virus 1 and bovine herpes virus 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
DETAILED DESCRIPTION OF THE INVENTION
(15) The present invention relates generally to the field of live and modified live, conventional and recombinant, vaccines. For example, compositions and methods are described that are useful for priming protective immunity in the presence of pre-existing maternal antibody. In some embodiments, simultaneously masking vaccines to avoid antibody neutralization while targeting those vaccines to specific cell types elicits an enhanced immune response. In other embodiments, the efficacy of the immune responses may be further enhanced using vectors that recruit and activate specific antigen-presenting cells.
I. Bovine Viral Diarrhea Virus
(16) Bovine Viral Diarrhea Virus (BVDV) is a leading cause of enteric, respiratory, and reproductive diseases in cattle, which results in significant economic losses in the cattle industry (1). BVDV is a small, enveloped, single-stranded RNA virus that is a member of the genus Pestivirus in the family Flaviviridae. Other closely related pestiviruses include Classical swine fever virus (CSFV) and ovine Border disease virus (BDV). The approximately 12.5 kb BVDV genome is flanked by 5′ and 3′ non-coding regions and consists of a single open reading frame (ORF) that encodes a 450 kDa precursor polyprotein that is processed by host and viral proteases to produce several structural and non-structural proteins (2, 3). The N.sup.pro, capsid protein, and glycoproteins E.sup.ms, E1, and E2, constitute the structural components of the virion, whereas NS2-3, NS4A-B, and NS5A-B constitute non-structural proteins (4-6). Resolution of acute BVDV infection requires cell-mediated immunity and significant portions of the N.sup.pro, capsid, E.sup.ms, E2, and NS2-3 antigens are highly conserved among BVDV strains and represent ideal targets for protection against multiple BVDV strains (32). The N.sup.pro, capsid, E.sup.ms, E2, and NS2-3 antigens contain B and T cell determinants that confer protection against infection with wt virus (20, 22, 23, 26-28). While several BVDV sub-unit vaccines based on these antigens have shown promising results, none of them have been used to date (41, 42, 83-85). BVDV is classified into genotypes 1 and 2 based on antigenic differences, as well as cytopathic (CP) and non-cytopathic (NCP) biotypes based on the presence or absence of cytopathic effect (CPE) in infected cell cultures (7-9). Classical BVDV infections cause severe diarrhea and respiratory disease, which is the most common cause of morbidity and mortality in feedyards (1). BVDV infections may also cause immunosuppression, abortion, stillbirth, congenital malformations, and the birth of persistently infected (PI) calves—the primary source of BVDV in cattle herds (7, 10, 11). A significant proportion of cattle entering feedyards are PI, and since a chute side test to identify these individuals is currently unavailable these calves pose a great risk to unprotected cattle (12, 13).
a) BVDV Immunity and Vaccines
(17) The initial control of BVDV infection in neonates and yearlings is conferred by colostrum-derived BVDV-specific neutralizing antibodies (14). The rate of neutralizing antibody decay in neonates correlates to antibody titer at the early age and is influenced by many factors. Consequently, a high risk of infection exists, especially in the presence of PI calves in the herd, when calves become vulnerable to BVDV as their neutralizing antibodies wane (14-16). While neutralizing antibodies provide protection against wt BVDV infection, CD4.sup.+ T cells are critical for clearance of infected cells (20, 21) due to their requirement in generating high affinity neutralizing antibody as well as optimal expansion and maintenance of CD8.sup.+ cytotoxic T cells (22). In addition to these “helper” roles, CD4.sup.+ T cells (as well as CD8.sup.+ T cells) can be directly cytotoxic towards BVDV infected cells. BVDV-specific CD4.sup.+ and CD8.sup.+ T cells have been shown to be present within protected animals (22, 23). Consequently, one embodiment of the present invention contemplates the development of vaccines capable of priming and expanding protective BVDV-specific effector/memory CD4.sup.+ and CD8.sup.+ T cell responses in neonates. Still further embodiments contemplate improved priming and expanding of protective BVDV-specific effector/memory CD4.sup.+ and CD8.sup.+ T cell responses in the presence of neutralizing antibody for uniform control of BVDV infection in calves and yearlings.
(18) Modified live BVDV and killed whole BVDV represent the primary vaccines available in the market. MLV vaccines are more efficacious than killed vaccines partly because the live virus infects vaccinates and replicates in vivo and thus delivers a more persistent antigen stimulus for B and T cell priming. Importantly, the virus generates high copies of its single-stranded RNA and the double-stranded intermediates which are ligands for TLR 7/8 and 3, respectively, and thus induce TLR-dependent pro-inflammatory responses that influence immune responses (73, 102, 103). MLV vaccines against BVDV also confer cross-protective immunity against different strains by inducing BVDV-specific neutralizing antibody, CD4.sup.+ and CD8.sup.+ T cells (18). Despite their ability to induce long lasting neutralizing antibodies and cell-mediated immunity, MLV vaccines have been known to cause mucosal disease, immunosuppression and in utero infections. Furthermore, MLV vaccines carry the risk of vaccine contamination with adventitious viruses and are ineffective in the presence of neutralizing antibodies (17, 82, 105).
(19) In contrast, killed (or inactivated) vaccines stimulate antibody responses that induce short-lived immunity, fail to induce cross-reactive immunity to different BVDV strains (19) and generate weak T-cell mediated responses that do not stimulate cytolytic T cells (106). Inactivated vaccines do not provide protection to fetal infections and tend to loose immunogenicity during preparation (107-109). Consequently, there is a need to generate alternative BVDV vaccines that are efficacious, safe, cost-effective, and non-susceptible to BVDV-specific antibody neutralization.
i) Antibody-Dependent BVDV Vaccine Inactivation
(20) MLV vaccines against BVDV prime protective antibody and T cell immunity but their efficacy is limited until neutralizing antibody levels have waned. MLV vaccines are more efficacious than killed BVDV vaccines partly because the live virus infects, vaccinates and replicates in vivo and thus delivers a more persistent antigen stimulus for B and T cell priming. However, in the presence of BVDV-specific neutralizing antibodies, as is the case in neonates with maternal antibodies, the antibody binds to the vaccine virus and antibody-mediated complement lysis and opsonization rapidly clears the virus, thus impairing effective priming (24, 25). In one embodiment, the present invention contemplates masking a MLV BVDV vaccine to protect it against complement lysis and thus allow targeting of the virus to the relevant antigen-presenting cells (APCs) for optimal induction of adaptive immunity.
ii) Protective BVDV Antigens
(21) The E2 and NS2-3 BVDV antigens are immunodominant, and neutralizing antibody as well as T cell responses directed against these antigens can confer protection against infection with wt virus (20, 22, 23, 26-28). The E2 envelope protein plays a major role in virus attachment and entry. Virus neutralization activity has been demonstrated predominantly for E2-specific antibodies, whereas the NS3 protein functions as a serine protease and RNA helicase; several B and T cell epitopes recognized by immune cattle have been identified (17, 26, 27, 29, 30). Cell-mediated immunity is required to resolve BVDV-infected cells and in vivo depletion of CD4.sup.+ T cells delays resolution of acute infection (20, 21). Importantly, evaluation of BVDV-specific immune responses following resolution of acute infection has revealed that the E2, NS2-3, capsid and N.sup.pro antigens are CD4.sup.+ T-cell determinants (23). In addition, major histocompatibility complex (MHC) DR-restricted T cell epitopes have been identified from conserved regions of E2 and NS2-3, and a large pool of potential class I allele-specific cytotoxic T cell peptide motifs from the BVDV polyprotein have been predicted (28, 31). Significant portions of the E2, NS2-3, N.sup.pro and capsid antigens are highly conserved among BVDV strains and represent potential targets for the development of T cell vaccines for protection against multiple BVD virus strains (32). In one embodiment, the present invention contemplates a vectored live vaccine expressing chimeric genes encoding protective BVDV antigens optimized for induction of robust effector/memory CD4.sup.+ and CD8.sup.+ T cell responses that will induce cell-mediated immunity in neonatal calves in the presence of neutralizing maternal antibodies.
II. Dendritic Cells
(22) Dendritic cells (DCs) have the unique ability to prime naïve T cells to become effector/memory T cells. Furthermore, DC antigen uptake can be influenced to enhance initiation of adaptive immunity. Flt3L and GM-CSF are molecular adjuvants (i.e. cytokines) that have been shown to enhance DC recruitment and vaccine efficacy in cattle, humans, and mice (69, 92, 93). Previous results have demonstrated that Flt3L and GM-CSF increase DC recndtment and significantly enhances antigen-specific CD4.sup.+ T cell responses and IFN-γ secretion in calves with diverse MHC class II haplotypes. A motif has been developed for directing high-affinity DC-vaccine interaction to enhance DC antigen uptake for optimal T cell priming. In addition, immunization of calves with a single dose of a DNA vaccine capable of DC recruitment, DC antigen targeting and DC activation, has been demonstrated to prime robust IFN-γ.sup.+ CD4.sup.+ T cell responses that undergo rapid recall upon boost. To improve priming of BVDV-specific adaptive immunity in neonates, one embodiment contemplates expanding previous achievements in DC recruitment, antigen targeting and activation to develop and evaluate the efficacy of the following contemporary BVDV vaccines: 1) A DC-targeted MLV BVDV vaccine capable of evading antibody neutralization; and 2) recombinant adenoviruses expressing protective BVDV antigen chimeras optimized for induction of robust CD4.sup.+ and CD8.sup.+ T cell responses. A central hypothesis to be examined with these BVDV vaccines is that immunization of neonatal calves with a single dose of a formulation containing adenoviruses expressing DC recruitment and activation factors, and a DC-targeted MLV BVDV vaccine capable of evading neutralizing antibodies or adenoviruses expressing DC-targeted chimeric BVDV antigens will prime and expand protective effector/memory CD4.sup.+ and CD8.sup.+ T cells.
(23) Langerhan cell (LC)-derived DCs preferentially activate cellular immunity and express high levels of CD205, which is involved in receptor-mediated antigen uptake through endocytosis (62-64). In one embodiment, priming of T cell immunity may be enhanced by targeting antigen to LCs. Importantly, antigens taken up by CD205 are presented by MHC class I and class II molecules, with CD205-targeted antigen presented at least 400 times more efficiently than free antigen (66). Surprisingly, targeting an antigen to CD205 using anti-CD205 antibody enhances systemic as well as mucosal antigen-specific CD4.sup.+ and CD8.sup.+ T cell responses by greater than 1000 fold in a mouse model (67).
III. Adenoviruses
a) Adenovirus Antigen Delivery System
(24) Replication-defective adenovirus vectors are attractive vaccine vehicles since they direct high transgene expression, are species-specific, and induce both innate and adaptive immune responses in mammalian hosts, in part, by DC modulation through Toll-Like Receptor (TLR)-dependent and -independent pathways (33-35). A single dose immunization with an adenovirus-vectored vaccine induces stronger cytotoxic T-cell responses than recombinant vaccinia virus vector, plasmid DNA, or a combination of the two (36). Importantly, adenovirus-vectored vaccines can be administered via multiple routes such as intradermal and intranasal for induction of systemic and mucosal immunity (37-39). In addition, adenovirus transduces LCs efficiently and immunization of neonates at birth with a single dose of an adenovirus-vectored vaccine induces robust immune responses (38, 40). Current adenovirus vectors are safe, can accommodate and express large transgenes, replicate at high titers in complementing cell lines, and their production is scalable and reproducible (34). Adenovirus vectors have been used to express BVDV antigens and to induce protective immunity in cattle (39, 41, 42). Replication-defective human adenovirus vectors are capable of inducing protective immunity in neonatal calves in the presence of high neutralizing antibodies, and a single dose of an adenovirus-vectored vaccine induces robust immune responses in neonates (33-35, 39, 41, 42). Furthermore, adenoviruses have been used to express cytokines for DC recruitment and activation (43, 44). In one embodiment, the present invention contemplates the use of adenoviruses expressing chimeras containing protective BVDV antigens optimized for induction of robust CD4.sup.+ and CD8.sup.+ T cell responses to induce cell-mediated immunity in neonatal calves in the presence of neutralizing maternal antibodies.
IV. Antigen Processing
a) Antigen Processing and Presentation
(25) Antigen processing and presentation are key events for induction, expansion and maintenance of antigen-specific T cells. DCs are rare but highly specialized professional APCs that have a unique ability to prime naïve T cells to become protective antigen-specific effector as well as memory T cells (45, 46). The mechanism of antigen uptake by DCs determines the type of T cell epitopes generated (47). Antigens from pathogens, such as BVDV (48), that infect DCs and access the cytoplasm are processed by the proteosome and are presented to CD8.sup.+ T cells by MHC class I molecules, whereas exogenous antigens are processed in the endosome/lysosome compartments and are presented to CD4.sup.+ T cells by MHC class II molecules (49, 50). DCs can also cross-present antigens from these processing pathways as well as antigens released from other cells (51, 52).
(26) In one embodiment, presentation of epitopes to T cells by DCs occurs in three distinct steps. First, maturing DCs in peripheral tissues function as sentinels, capturing antigen using specific receptors, phagocytosis or by uptake in the fluid phase, and then migrate to the draining lymphoid organs (45, 46). Antigen acquisition is followed by antigen processing and formation of peptide-MHC complexes. In the presence of sustained antigen uptake as well as supply of MHC molecules, these complexes progressively accumulate on DC surfaces (53, 54). The third step is T cell recognition whereby as few as 100 peptide-MHC complexes serially engage and trigger as many as 18,000 T cell receptors (55-57). Primed T cells then undergo clonal expansion to become circulating effectors, which are short-lived or memory cells ready to respond to recall antigen (58). In one embodiment, the present invention contemplates targeting a masked MLV BVDV vaccine or adenovirus-expressed protective BVDV antigen chimera to DCs in neonates to enhance priming of BVDV-specific T cells that will in turn provide quality help to B cells for optimal antibody responses and improved vaccine efficacy.
V. Targeting and Masking
a) Targeting High-Affinity DC-BVDV Antigen Interaction to Enhance Priming
(27) DCs are the key APCs required for optimal induction of a primary immune response following vaccination—not macrophages or B cells. However, the frequency of DCs in tissues is less than 1% of all nucleated cells (59-61). Consequently, a vaccine that contains factors for DC recruitment and a motif that directs efficient targeting of a “masked” MLV BVDV vaccine or adenovirus-expressed BVDV antigens to a DC antigen receptor with high affinity may significantly increase the amount of antigen taken up by DCs as well as the number of DCs processing and presenting BVDV antigens, thus enhancing priming of BVDV-specific T cells in neonates with neutralizing antibodies.
(28) Maturing LC-derived DCs in the skin express high levels of CD205, which is involved in receptor-mediated antigen uptake through endocytosis (62). LC-derived DCs are confined to the T cell-rich zone in the draining lymph nodes where they preferentially activate cellular immunity, whereas dermal DCs are located just beneath the B cell follicles and appear to preferentially activate humoral immunity (63-65) and thus, antigen targeting to LCs should enhance priming of effector/memory T cell immunity. Importantly, antigens taken up by CD205 are processed and presented by MHC class I and class II molecules, and a CD205-targeted antigen is presented at least 400 times more efficiently than free antigen (66). Surprisingly, targeting an antigen to CD205 using anti-CD205 antibody enhances systemic as well as mucosal antigen-specific CD4.sup.+ and CD8.sup.+ T cell responses by >1000 fold in a mouse model (67). Furthermore, targeting an antigen to maturing, but not mature, DCs renders non-immunogenic antigens immunogenic and induces significant protective cellular immunity in a mouse model (68). Previous results demonstrate that Flt3L and GM-CSF increases DC recruitment to the intradermal immunization site (
b) Activation of BVDV Antigen-Loaded DCs to Enhance Priming
(29) Naive T cells require two distinct signals from APCs for proper activation and induction of differentiation: the first signal is provided by peptide antigens in the context of MHC molecules, the second signal is delivered by co-stimulatory molecules such as CD80 or CD86 present on DCs (71). For antigen-loaded DCs to provide these signals effectively, they require activation to up-regulate surface expression of MHC-peptide complexes and co-stimulatory molecules, and to secrete pro-inflammatory molecules such as IL-12 (72). DC activation is an innate response that adjuvants as well as live vaccines stimulate through TLR signaling, chemokine and cytokine secretion (73). Expression of CD80/CD86 is upregulated by TLR ligands, TNF-α and IFN-γ as well as interaction between CD40 on DCs and CD40 ligand (CD40L) expressed by activated T lymphocytes (71-73). However, DCs from CD40.sup.−/− and CD40L.sup.−/− mice do not elicit CD4.sup.+ and CD8.sup.+ T cell immunity, even though the DCs present antigen on MHC class I and II molecules and express high levels of CD80/86 (72). A distinct CD40/CD40L signal that functions together with antigen presentation and co-stimulation is required to generate functional CD4.sup.+ T helper and CD8.sup.+ CTLs (72). This signaling critically requires APC-T cell contact, local CD40L secretion, or an agonistic anti-CD40 antibody (67, 74, 75). Furthermore, this signaling results in increased production of pro-inflammatory cytokines such as IL-12, which is a powerful inducer of IFN-γ production and Th1 differentiation (76). More importantly, DC activation through CD40 signaling overcomes tolerance and may release immature DCs from the control of regulatory CD4.sup.+ CD25.sup.+ T cells (77). In one embodiment of the present invention, immunization of neonatal calves with a single dose of a formulation containing adenoviruses expressing Flt3L/GM-CSF for DC recruitment, CD40L for DC activation, mixed with a DC-targeted MLV BVDV vaccine capable of evading neutralizing antibodies or adenoviruses expressing DC-targeted chimeric BVDV antigens induces protective CD4.sup.+ and CD8.sup.+ T cell immunity in the presence of neutralizing antibodies.
c) In Vivo Enhancement of BVDV-Specific T Cell Immunity in Neonates
(30) Heightened and improved protection of neonatal calves and yearlings against BVDV infection requires effective generation and preservation of BVDV-specific immunological memory. The potential of a vaccine formulation containing adenoviruses expressing Flt3L/GM-CSF/CD40L mixed with a DC-targeted “masked” MLV BVDV vaccine or adenoviruses expressing DC-targeted chimeric BVDV antigens to enhance priming and expansion of protective BVDV-specific CD4.sup.+ and CD8.sup.+ T cell responses is evaluated in neonatal calves with protective titers of BVDV neutralizing antibodies. More importantly, the efficacy of such BVDV vaccine formulations to confer protection is evaluated upon challenge following decline of passive immunity.
(31) Immunization of young seronegative calves with a single dose of a MLV vaccine against BVDV confers protection and thus neonates are immune competent (75). A MLV vaccine is very susceptible to neutralization by antibodies but the vaccine can be masked to create a coat around the virion; that coat may be modified to contain a motif(s) for targeting the vaccine to DCs. As depicted in
(32) Although MLV vaccines against BVDV are efficacious in adults, they are known to cause mucosal disease, immunosuppression, in utero infection and carry the risk of vaccine contamination with adventitious viruses (17, 82). In an attempt to develop improved vaccines, several recombinant protein, DNA, and live-vectored BVDV sub-unit vaccines have been generated using the genes encoding the E2, NS2-3, capsid, and N.sup.PRO antigens; however there is no commercially available subunit vaccine against BVDV (41, 42, 83-85). Recombinant DNA and live-vectored BVDV subunit vaccines can overcome neutralizing maternal antibodies and induce T cell immunity in neonatal calves. However, a recombinant live vaccine with a comprehensive representation of protective B and T cell determinants from BVDV 1 and 2 has yet to be developed and tested in neonatal calves. In one embodiment, the present invention contemplates developing a contemporary vaccine against BVDV using a chimeric gene generated by fusing the DNA sequences encoding hydrophilic domains of consensus amino acid sequences generated by aligning the N.sup.pro, capsid, E.sup.ms, E1, and E2 polypeptide sequences of sequenced BVDV 1 and 2 genomes. This alignment generates a mosaic chimera that captures the antigen repertoire of currently known BVDV isolates. In a preferred embodiment of the present invention, immunization with the mosaic chimeric antigen confers broad protection against BVDV 1 and 2. In a preferred embodiment, this chimera may be modified to contain the sequences encoding the CC98 DC-targeting motif (
(33) In one embodiment, the present invention contemplates inducing broad cell-mediated immunity in neonatal calves in the presence of neutralizing maternal antibodies by immunizing neonatal calves with a single dose of a vaccine formulation containing adenoviruses expressing Flt3L/GM-CSF/CD40L mixed with 1) a DC-targeted MLV BVDV vaccine capable of evading neutralizing antibodies, or 2) an adenoviruses expressing DC-targeted mosaic BVDV antigens optimized for induction of robust effector/memory CD4.sup.+ and CD8.sup.+ T cell responses.
Experimental
(34) The following are examples that further illustrate embodiments contemplated by the present invention. It is not intended that these examples provide any limitations on the present invention. In the experimental disclosure that follows, the following abbreviations apply: eq. or eqs. (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmoles (picomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanogram); vol (volume); w/v (weight to volume); v/v (volume to volume); L (liters); ml (milliliters);. μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); C (degrees Centigrade); rpm (revolutions per minute); DNA (deoxyribonucleic acid); kDa (kilodaltons); kb (kilobase).
(35) Effective control and long-term containment of BVDV requires the development of vaccines capable of stimulating robust effector/memory B and T cell immunity in neonatal calves and yearlings. An optimal vaccine should induce immunity in neonates that are not adequately protected by colostrum-derived antibodies thereby avoiding the period of vulnerability to infection as passive immunity wanes. Results described herein extend the present inventors previous achievements in bovine DC recruitment, antigen targeting and activation to develop contemporary BVDV vaccines for effective priming of BVDV-specific adaptive immunity in neonates in the presence of neutralizing antibodies. In one embodiment, the vaccine optimization strategies disclosed herein augment BVDV vaccine immunogenicity at a single DC level. In a preferred embodiment, BVDV vaccine immunogenicity is augmented by employing: 1) Flt3L/GM-CSF to attract DCs to the immunization site, 2) CD40L (and the adenovirus vector itself) to provide potent TLR-dependent and -independent DC activation, and 3) the CC98-348 diabody to protect and target the MLV vaccine against BVDV 1 and 2 to DCs; wherein the CC98 scFv motif provides the specificity required for DC antigen targeting of protective BVDV antigens.
I. Bi-Specific Diabody Production
a) Monoclonal Antibody Production
(36) Monoclonal antibody (mAb) production involves a series of steps known to those skilled in the art, including (for example) antigen isolation, lymphocyte stimulation, myeloma cell fusion, identification of mAb-secreting cells and characterization of mAb specificity and affinity. Briefly, antibody producing lymphocytes are stimulated by in vitro or in vivo by immunization of an animal with a preparation of a desired antigen, including for example, BVDV 1 and 2 antigens such as N.sup.pro, capsid, E.sup.ms-E1, E2, NS2 and NS3. Spleens are removed from the immunized animals 72 hours following the last antigen boost and placed in Dulbecco's minimal essential medium (DMEM) supplemented with 4.5 gm/L of glucose 1000 U/ml of penicillin and 100 μg/ml streptomycin. The spleens are teased apart with 25 gauge needles and the splenocytes are washed three times with DMEM prior to being resuspended at a constant concentration in DMEM. In general approximately 100 million cells are obtained from each spleen. The splenocyte suspension is then mixed with a continuously proliferating myeloma cell line in 96 well microtiter plates to generate cell fusions. The myeloma cells are sensitive to hypoxanthine-aminopterin-thymidine (HAT) medium by virtue of their lacking enzymes such as thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyl transferase (HGPRT). This allows selection of hybrids to be accomplished by growth in HAT medium. Myeloma cell lines utilized for cell fusions may be derived from a BALB/c mouse MOPC 21 myeloma as described by Kholer et al, Eur. J. Immunol., Vol. 6, pp. 292-295 (1976). Fused cells are grown in DMEM supplemented with 4.5 gm/l glucose, 20 mm glutamine, 1000 U/ml penicillin, 100 μg/ml streptomycin and 20% fetal calf serum (complete medium).
(37) Microtiter wells are screened for cell proliferation 10 to 20 days following cell fusion. The mAbs secreted by individual clones are assayed for their ability to bind to the target antigen using assays such as ELISA, antigen microarray assay or immuno-dot blot. Hybridoma cells identified as positive secretors of the desired mAb are serially passaged to establish continuously proliferating cell lines with relatively stable genetic constitutions. These cell lines or clones are propagated indefinitely in tissue culture or in vivo in syngeneic or immunocompromised hosts where they continue to synthesize and secrete antibody to the hepatitis viral antigens. The remaining cells are frozen and stored under liquid nitrogen.
(38) Three separate assays may be used to screen mAbs secreted from individual clones for BVDV antigen specificity and affinity. In the first phase, one hundred and twenty μl of culture supernatant is removed from the microtiter plates and diluted to 200 μl with complete medium. Antigen coated beads are incubated for 24 hours at room temperature followed by extensive washing with distilled water. .sup.125I-labeled antigen is then added (100-150,000 cpm) and the plates are incubated at room temperature for an additional 36 hr. period. The beads are then extensively washed with distilled water and counted in a Packard gamma counter. The second phase is a solid phase radioimmunoassay affinity purified goat anti-mouse F(ab′).sub.2 as described by Williams et al., Cell, Vol. 12, pp. 663-673 (1977). The third phase utilizes a microhemagglutination reaction to evaluate the mAb's ability to agglutinate antigen coated human 0-negative red blood cells as described by Wands et al., Gastroenterology, Vol. 68, pp. 105-112 (1975).
(39) Seven BVDV-specific mAb clones have been generated that neutralize both BVDV 1 and 2 in vitro (clones 2C11, 1C2, 2D7, 2H5, 1G4, 1A4, 2F12, data not shown). These mAb clones are currently undergoing a second round of cloning; once the sub-clones have been tested for BVDV neutralization, at least one mAb will be selected, characterized and the hybridoma secreting the mAb will be used to generate single chain antibody.
b) Single Chain Variable Fragment (scFv) Production
(40) A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (V.sub.H) and light chains (V.sub.L) of immunoglobulins, connected with a short linker peptide, typically of ten to about 25 amino acids. In some embodiments the linker is rich in glycine for flexibility, as well as serine or threonine for solubility, and can connect the N-terminus of the V.sub.H with the C-terminus of the V.sub.L, or vice versa. scFv proteins retain the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. scFv fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites used to purify antibodies.
(41) A variety of methods for producing a scFv that targets a specific antigen are known to those skilled in the art. A first method requires generating a mouse hybridoma clone (using the methods above) followed by isolating cDNA from the hybridoma and then amplifying the genes encoding the variable heavy (V.sub.H) and variable light chains (V.sub.L) of the immunoglobulin by PCR. The gene encoding the V.sub.H and the V.sub.L chains are then fused in-frame to create one open-reading frame. Nucleotides encoding a peptide linker are incorporated between the genes encoding the V.sub.H and the V.sub.L chains for flexibility and the resultant chimeric gene is sub-cloned into a protein expression vector (for example, bacterial expression vectors or eukaryotic expression vectors such as yeast) for the scFv production. A second method requires creating a scFv phage display library using cDNA from a hybridoma secreting a specific mAb, which is then screened with an antigen of interest. A third method is to screen a premade scFv antibody phage display library with an antigen of interest to identify scFv clones directly.
(42) Divalent (or bivalent) single-chain variable fragments may be engineered by linking two scFvs. In one embodiment, this may be accomplished by producing a single peptide chain with two V.sub.H and two V.sub.L regions, yielding tandem scFvs. In another embodiment, scFvs are generated with linker peptides that are too short for the two variable regions to fold together (about five amino acids), thereby forcing the scFvs to dimerize to form a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Consequently, diabody drugs may be administered at much lower concentrations than other therapeutic antibodies while remaining capable of highly specific antigen targeting in vivo.
II. DC Recruitment
(43) Flt3L and GM-CSF have been shown to increase DC recruitment to the intradermal immunization site. Quantitative immunohistochemistry using anti-CD1 mAb TH97A demonstrates that Flt3L and GM-CSF enhance DC recruitment to the skin (
III. Binding of Bovine CD205-Specific rscFv to Skin DCs
(44)
IV. Immunization of Neonatal Calves with a DNA Vaccine
(45) Results demonstrate that immunization of calves with a single dose of a DNA vaccine capable of DC recruitment, CD205 antigen targeting and CD40L-directed DC activation was able to prime and expand robust IFN-γ.sup.+ CD4.sup.+ T cell responses that undergo rapid recall upon boost (70).
V. Cytokine Expression by Recombinant Adenoviruses
(46) Recombinant adenoviruses encoding bovine Flt3L, GM-CSF, or CD40L were generated using ViraPower Adenoviral Gateway Expression Kit (Invitrogen) according to the manufacturer's instructions.
VI. Generation of Bovine CD205-Specific scFv
(47) A recombinant bovine CD205-specific scFv for DC-targeting and a scFv from an isotype-matched control mAb were generated in 293 Free-style cells (Invitrogen). The scFv is a mouse IgG (V.sub.H-V.sub.L chimera) but most of the amino acids in the V.sub.H chain and the whole V.sub.L chain may be replaced with bovine IgG residues without compromising function (89). Briefly, the ORFs of the V.sub.H and V.sub.L chains of the mAb CC98 (IgG2b) were amplified from the CC98 hybridoma (90) cDNA by separate asymmetric PCR (91) using a Mouse ScFv Module (Amersham) according to the manufacturer's instructions.
(48) The asymmetric PCR products were joined by overlap extension PCR (91), resulting in the CC98 scFv containing the (G.sub.4S).sub.3 (SEQ ID NO: 4) linker between the V.sub.H and the V.sub.L.
(49) Similarly, a control scFv, designated IC scFv, was constructed using cDNA generated from a hybridoma that secretes an isotype-matched mouse IgG2b mAb. To test CD205 binding, FLAG-tagged recombinant CC98 scFv, a rCC98 scFv fused to a 30 kDa protein (MSP1), and the rIC scFv control was expressed in 293 Free-Style cells (Invitrogen) and affinity purified using anti-FLAG agarose resin (Sigma).
(50) Binding of FLAG-tagged rCC98 scFv to bovine CD205 receptor was tested by immunocytochemistry using 293-F cells transfected with a construct (pCD205) encoding bovine CD205 receptor (90) (
VII. Generation of the CC98-348 Bi-Specific Diabody
(51) A diabody (designated CC98-348) containing the CD205-specific CC98 scFv fused in-frame to a BVDV E2 antigen-specific scFv (generated from mAb 348) that neutralizes BVDV 1 and 2 (79) was generated to both mask MLV vaccines against BVDV 1 and 2 and target DCs. In addition to mAb 348, other BVDV neutralizing mAbs are currently being developed to allow the production of additional bi-specific diabodies. Several promising clones are currently being screened for neutralization of BVDV1&2 (i.e. clones 2C11, 1C2, 2D7, 2H5, 1G4, 1A4, 2F12).
(52)
(53) The N-terminal CD5 secretory signal sequence, arrangement of the variable domains as well as the linker sequences A (G.sub.4S) (SEQ ID NO: 2) and B (G.sub.4SG.sub.2RASG.sub.4SG.sub.4S) (SEQ ID NO: 3), and the C-terminal FLAG tag are shown.
(54) The structure of the negative control diabody (designated IC-348) is similar but with V.sub.H and V.sub.L domains from an isotype-matched control scFv. A second negative control diabody for the 348 scFv (designated IC-IR) was also generated.
(55) The V.sub.H and V.sub.L fragments of the CC98 scFv and the cDNA encoding the mAb 348 (79) were amplified by separate asymmetric PCR (91) using a Mouse ScFv Module (Amersham). The asymmetric PCR products were joined in-frame by overlap extension PCR (91) in the format V.sub.HCC98.sub.linkerAV.sub.L348.sub.linkerBV.sub.H348.sub.linkerAV.sub.LCC98 (
(56) This chimeric gene was modified to contain a 5′ linked eukaryotic secretory signal (designated CD5ss) (69, 86) and a 3′ linked sequence encoding a FLAG-tag. The resultant chimeric gene (designated cd5cc98-348flag) was used to generate a recombinant adenovirus (designated AdCC98-348) for expression of recombinant CC98-348 in 293 Free-style cells (Invitrogen). Expression of the recombinant CC98-348 diabody was verified by immunocytochemistry using anti-FLAG mAb (Sigma) and the protein is affinity purified using anti-FLAG agarose gel (Sigma). Similarly, the V.sub.H and V.sub.L fragments from the IC control scFv were fused in-frame with the 348 scFv or an irrelevant scFv to generate control diabodies (designated IC-348 and IC-IR, respectively). Binding of the CC98-348 diabody to bovine CD205 was verified by immunocytometric analysis of 293 F cells transfected with pCD205. CC98 mAb is the positive control whereas the IC-348 and the IC-IR diabodies serve as negative controls. Binding of the CC98-348 and the IC-348 diabodies to BVDV was verified by ELISA. The mAb 348 served as a positive control, whereas an isotype-matched mAb and the IC-IR diabody served as negative controls.
VIII. Generation of Adenoviruses Expressing BVDV Antigens
(57) Resolution of acute BVDV infection requires cell-mediated immunity. The N.sup.pro, capsid, E.sup.ms, E2, and NS2-3 antigens of BVDV contain B and T cell determinants that confer protection against infection with wt virus (20, 22, 23, 26-28). Since significant portions of the N.sup.pro, capsid, E.sup.ms, E2, and NS2-3 antigens are highly conserved among BVDV strains they represent ideal targets for protection against multiple BVDV strains (32).
(58) Recombinant adenoviruses expressing chimeric BVDV antigens will be generated using the ViraPower Adenoviral Gateway Expression Kit (Invitrogen). This vector is replication-incompetent in non-complementing cell lines due to deletion of the E1 and E3 genes but replicates to 10.sup.12 pfu/ml in complementing cells (33). The vector can accommodate up to 7.5 kb of foreign DNA and utilizes the human CVM promoter to drive high level transgene expression (33). Hydrophilic domains of the N.sup.pro-capsid (amino acids 1-252), E.sup.ms-E1 (amino acid 270-564), and the E2 antigen (amino acids 693-1035) from currently sequenced BVDV type 1 or type 2 were aligned and the consensus amino acid sequences used to design broadly representative mosaic chimeric polypeptides, designated BVDV1.sub.NcapE2 and BVDV2.sub.NcapE2, respectively. The mosaic chimeric polypeptides sequences have been used to generate codon-optimized synthetic genes (for optimal protein expression in bovine cells in vivo) and these genes have been modified to contain the gene encoding the CC98 scFv for DC-targeting at the 5′ end the gene encoding FLAG tag at the 3′ end (69, 86) Initial results from expression constructs (DNA and adenovirus) bearing the chimeric genes demonstrate the ability to express CD205-targeted antigen; experiments to optimize protein expression are currently being performed. The resultant chimeric genes have been used to generate recombinant adenoviruses, designated AdCC98-BVDV1.sub.NcapE2 and AdCC98-BVDV2.sub.NcapE2, respectively.
(59) The CD5 secretory signal sequence, arrangement of the CC98 variable domains, linker sequences A (G.sub.4S).sub.3(SEQ ID NO: 4) and B (G.sub.4S) (SEQ ID NO: 2), hydrophilic domains of N.sup.pro, capsid, E.sup.ms-E1, E2, and the FLAG tag are shown.
(60) The structure of the control chimeras, IC-BVDV1.sub.NcapE2 and IC-BVDV2.sub.NcapE2, are similar but with the IC variable domains. Similarly, an adenovirus, designated AdCC98-BVDV1.sub.NS2-3 and AdCC98-BVDV2.sub.NS2-3, respectively, have been generated using the hydrophilic domains of the NS2-3 antigens (amino acids 1300-2384).
(61) Protein expression was verified by immunocytochemistry using anti-FLAG mAb (Sigma) and BVDV-specific mAbs and polyclonal antibodies. CD205 binding was verified by immunocytometric analysis of 293A cells transfected with the pCD205 construct. Processing and presentation of the antigens expressed by these adenoviruses was verified by .sup.3H-thymidine incorporation and cell cytotoxicity using BVDV-reactive PBMCs.
IX. Protection and DC Targeting of the MLV BVDV Vaccine
(62) Efficacy of MLV BVDV vaccine virus protection against neutralization by BVDV-specific antibodies and DC targeting by the CC98-348 diabody was evaluated in vitro. To evaluate efficacy of the CC98-348 diabody to protect the BVD virus from complement-mediated lysis, 4×10.sup.6 TCID.sub.50 of a commercial MLV vaccine against BVDV 1 and 2 was incubated at 37° C. for 1 hr with either the CC98-348 or IC-348 diabodies in the presence or absence of BVDV-specific neutralizing antibodies in medium containing non-inactivated bovine serum to preserve complement activity. The mixture was then added to 293A cell monolayers (BVD virus does not infect 293A cells) transfected with bovine CD205 or non-transfected cells as controls. The monolayers were incubated at 37° C. for 1 hr to allow binding of the CC98 to CD205, washed to remove unbound complexes and pH 3.5 growth medium was added for 10 minutes to free the diabody-protected virus bound to CD205 transfected 293A cells. The medium, free of 293A cells, was recovered and added to MDBK cell monolayers to support BVDV replication and the pH was adjusted to 7.4. The MDBK monolayers were incubated for 6 hrs, washed, and normal growth medium added. After three days the presence of BVDV in the MDBK cells was evaluated by immunocytometric analysis using mAb 348 (94). In one embodiment of the present invention, it is expected that incubation of BVDV with CC98-348 or IC-348 diabody, but not with BVDV neutralizing antibodies, will result in protection of BVD virus but only CC98-348 is expected to protect and target the virus to CD205 as evidenced by the detection of virus in MDBK cells.
X. CC98-348 Mediated Targeting, Binding and Protection
(63) The ability to demonstrate that the CC98-348 conjugate targets BVDV to CD205 and inhibits binding of a FITC-labeled anti-BVDV polyclonal antibody is critical evidence for proof of concept. Results demonstrate that the CD205-BVDV E2-specific diabody coat MLV vaccine virions and protect the vaccine from antibody-mediated complement lysis while also targeting the coated virions to DCs. In one embodiment of the present invention, this bi-functional capability dramatically improves the efficacy of current BVDV MLV vaccines in neonatal calves.
(64) The bi-specific CC98-348 diabody consists of the bovine CD205-specific mAb CC98 conjugated to the BVDV neutralizing mAb 348 (Deregt, D., et al 1998. Virus Research 53:81-90).
(65) The ability of the CC98-348 conjugate to bind to CD205 was demonstrated by immunocytometric analysis of cells that express CD205 (
(66) The ability to target BVDV to CD205 by the CC98-348 conjugate was also demonstrated by immunocytochemistry (
(67) To test whether the CC98-348 conjugate protects BVDV from binding of anti-BVDV polyclonal antibody, CD205 transfected 293A cells were treated as in A above and following extensive washes, FITC-labeled anti-BVDV polyclonal antibody was added and the cells evaluated by FACS. Binding of the CC98-348 conjugate to BVDV completely inhibited binding of the FITC-labeled anti-BVDV polyclonal antibody (Data not shown). To test whether the CC98-348 conjugate protects the BVDV from anti-BVDV antibody-directed complement lysis, BVDV was masked using the CC98-348 conjugate and the mixture subjected to rabbit complement in the presence of anti-BVDV polyclonal antibody. BVDV mixed with negative control conjugate, CC98, or 348 served as the controls. This assay is not critical since we have shown that the CC98-348 conjugate completely inhibited binding of anti-BVDV polyclonal antibody to BVDV.
XI. Protective Immunity in Neonatal Calves Vaccinated with an Adenovirus Expressing Flt3L/GM-CSF/CD40L Mixed with a DC-Targeted MLV Vaccine Against BVDV
(68) One embodiment of the present invention contemplates priming protective immunity in neonatal calves by immunizing with a single dose of adenoviruses expressing Flt3L/GM-CSF/CD40L mixed with a DC-targeted MLV vaccine against BVDV 1 and 2.
(69) We have generated a bovine CD205-specific scFv (designated CC98) for DC-antigen targeting and an isotype-matched control scFv (
(70) Generation of functional CD4.sup.+ T helper and CD8.sup.+ CTLs requires DC activation through CD40 signaling and increases production of pro-inflammatory cytokines and Th1 differentiation (67, 74, 75) (76). Importantly, DC activation through CD40 signaling overcomes tolerance and may release immature DCs from the control of regulatory CD4.sup.+ CD25.sup.+ T cells (77). The recombinant adenovirus, AdCD40L, expressing bovine CD40L (
a) Immunization of Neonatal Calves
(71) Five groups (n=5) of age-matched neonatal calves with protective BVDV neutralizing antibody titers were used in the present study (15). A herd of greater than 6,000 animals comprising young (1 week to 4 months old) Holstein calves (StoneyPoint AgriCorp, Melissa, Tex.) was used to select age-matched calves following screening for BVDV neutralizing antibody titers as previously described (97). Group size was estimated using IFN-γ.sup.+ CD4.sup.+ T cell response data from calves primed with a single dose of a DNA vaccine capable of DC recruitment, CD205 antigen targeting and CD40L-directed DC activation. The treatment group for that study was composed of DRB3*1101-matched calves (n=5) immunized with a single dose of the CC98MSP1 DNA vaccine (0.25 mg) encoding a DC-targeted A. marginale MSP1 antigen mixed with DNA constructs expressing FlT3L/GM-CSF/CD40L (0.5 mg each) (
(72) Each calf in group A was inoculated intradermally with 5×10.sup.9 TCID.sub.50 of the CC98-348.sub.BVDV vaccine mixed with the AdFlt3L/AdGM-CSF/AdCD40L (5×10.sup.9 pfu/each). The 5×10.sup.9 pfu dose of a recombinant adenovirus has been shown to be effective in cattle (98). Each calf in groups B and C similarly inoculated with the IC-348.sub.BVDV vaccine or with a non-protected and non-targeted BVDV vaccine (IC-IR.sub.BVDV), respectively. Calves in group D are inoculated with the CC98-348.sub.BVDV vaccine, as in group A, but mixed with the Adluciferase virus to serve as a control for the cytokines. Negative control calves in group E were inoculated with the cytokine adenoviruses, as in group A, but mixed with an equivalent amount of the CC98-348 diabody used in the CC98-348.sub.BVDV vaccine.
b) Analysis of Immune Responses
(73) Following immunization, peripheral blood mononuclear cells (PBMCs) were used to evaluate T cell responses, whereas serum used to evaluate antibody responses and virus neutralization. BVDV-specific IFN-γ.sup.+ CD4.sup.+ and IFN-γ.sup.+ CD8.sup.+ T cells, CD4.sup.+ T cell proliferation, CD8.sup.+ T cell cytotoxicity and antibody responses induced by the vaccine immunization was evaluated by IFN-γ.sup.+ ELISPOT, .sup.3H-thymidine incorporation, cell-mediated cytotoxicity assay (using autologous BVDV 1- or 2-infected ConA stimulated lymphoblasts as targets), ELISA, and BVDV neutralization assays, respectively, as previously described (22, 97, 99). These parameters were tested bi-weekly to determine the post-vaccination interval required to detect statistically significant BVDV-specific immune responses. The significance of any differences in BVDV-specific immune responses between treatment group A (Ad-cytokines/CC98-348.sub.BVDV) and the control groups B (Ad-cytokines/IC-348.sub.BVDV); C (Ad-cytokines/IC-IR.sub.BVDV); D (Adluc/CC98-348.sub.BVDV); and E (Ad-cytokines/CC98-348) was analyzed using ANOVA.
c) Analysis of Recall Responses and Protection
(74) Following the decline of neutralizing antibodies to below protective titers in the negative control calves (group E), which occur at approximately 6-8 months (15), live challenge was used to determine efficacy of the Ad-cytokine/CC98-348.sub.BVDV vaccine. All calves was challenged by intranasal inoculation with 5×10.sup.6 TCID.sub.50 of BVDV (NY-1 non-cytopathic strain) and then evaluated for clinical and hematological parameters, and viremia as previously described (97). Any reactions to the challenge were calculated as a disease reaction index based on a previously described combination of viremia, clinical and hematological parameters (100). One-week post challenge, BVDV-specific antibody titers, virus neutralization, and T cell responses was evaluated. The significance of the differences in BVDV-specific immune responses and disease indices between treatment group A (Ad-cytokines/CC98-348.sub.BVDV) and the control groups B (Ad-cytokines/IC-348.sub.BVDV); C (Ad-cytokines/IC-IR.sub.BVDV); D (Adluc/CC98-348.sub.BVDV) and E (Ad-cytokines/CC98-348) was analyzed using ANOVA.
XII. Protective Immunity in Neonatal Calves Vaccinated with an Adenovirus Expressing Flt3L/GM-CSF/CD40L Mixed with DC-Targeted BVDV Antigens
(75) There is a need for alternative BVDV vaccines that are efficacious, safe, cost-effective, and non-susceptible to BVDV-specific antibody neutralization. One embodiment of the present invention contemplates priming protective immunity in neonatal calves by immunizing with a single dose of adenoviruses expressing Flt3L/GM-CSF/CD40L (for DC recruitment and activation) and DC-targeted protective BVDV antigens. In one embodiment, the immunization induces robust effector/memory CD4.sup.+ and CD8.sup.+ T cell responses to prime and expand cell-mediated immunity in the presence of neutralizing antibodies.
a) Immunization of Neonatal Calves
(76) Four groups (n=5) of age-matched neonatal calves with protective colostrum-derived BVDV neutralizing antibody titers were used in this study. Group size was estimated using MSP1-specific IFN-γ.sup.+ CD4.sup.+ T cell response data (
(77) Briefly, calves in groups A and C will be inoculated intradermally with a single dose of the adenoviruses expressing the DC-targeted BVDV antigens (5×10.sup.9 pfu/each) mixed with either the adenoviruses expressing the cytokines (5×10.sup.9 pfu/each) or the Adluciferase control, respectively. Calves in group B will be inoculated similarly, but with a mixture of the adenoviruses expressing the non-targeted BVDV antigens mixed with the adenoviruses expressing the cytokines. Calves in group D will receive the Adluciferase mixed with the adenoviruses expressing the cytokines.
b) Analysis of Immune Responses
(78) Following immunization, PBMCs were used to evaluate T cell responses and sera was used to evaluate antibody responses and virus neutralization. BVDV-specific IFN-γ.sup.+ CD4.sup.+ and IFN-γ.sup.+ CD8.sup.+ T cells, CD4.sup.+ T cell proliferation, CD8.sup.+ T cell cytotoxicity and antibody responses was evaluated by IFN-γ.sup.+ ELISPOT, .sup.3H-thymidine incorporation, cell-mediated cytotoxicity assay, ELISA, and BVDV neutralization assays, respectively. These parameters were tested bi-weekly to determine the post-vaccination interval required to detect statistically significant BVDV-specific immune responses. The significance of the differences in BVDV-specific immune responses between treatment (group A) and the controls (groups B-D) was analyzed using ANOVA.
c) Analysis of Recall Responses and Protection
(79) Following decline of neutralizing antibodies to below protective titers (6-8 months) in the negative control calves (group D), live challenge was used to determine the efficacy of the vaccine formulation containing the adenoviruses expressing the DC-targeted BVDV antigens and the cytokines. All calves were challenged as described above. Disease indices, BVDV-specific antibody titers, virus neutralization, and T cell responses were evaluated as described above. The significance of the differences in disease indices and BVDV-specific immune responses between the treatment (group A) and the controls (groups B-D) was analyzed using ANOVA.
XIII. Potential Outcomes and Alternative Strategies
a) BVDV Vaccine Protection and DC Targeting
(80) In one embodiment, the present invention contemplates testing whether CC98-348 diabody-dependent BVDV vaccine protection and DC targeting, and the AdFlt3L/AdGM-CSF/AdCD40L-directed DC recruitment and activation, significantly enhances priming and expansion of BVDV-specific B and T cell responses in the presence of neutralizing antibodies and confers protection upon challenge.
(81) The AdFlt3L/AdGM-CSF/AdCD40L viruses efficiently express functional cytokines (
(82) The CC98 scFv binds and targets antigen to bovine CD205 in vitro and to DCs in situ (
(83) If, after one immunization, a significant BVDV-specific immune responses and/or protection is detected in group A (Ad-cytokines/CC98-348.sub.BVDV) but none in group B (Ad-cytokines/IC-348.sub.BVDV), the hypothesis that intradermal inoculation of neonatal calves with a single dose of a vaccine formulation containing adenovirus expressing Flt3L/GM-CSF/CD40L mixed with CC98-348-coated MLV vaccine against BVDV 1 and 2 is capable of priming and expanding protective BVDV-specific immune responses in the presence of neutralizing antibody will be accepted. The hypothesis will also be accepted if, after one immunization, we detect significantly greater BVDV-specific immune responses and/or protection in group A as compared to groups B-E. Failure to detect significant BVDV-specific immune responses in group A after a single immunization will indicate that immunization of neonatal calves with a single dose of the Ad-cytokines/CC98-348.sub.BVDV vaccine is not sufficient to prime and expand significant BVDV-specific immune responses in the presence of neutralizing antibodies and a booster dose is required. Detection of a rapid recall response and/or significant protection after challenge in group A as compared to all the other groups (B-E), will indicate that immunization of neonatal calves with a single dose of the Ad-cytokines/CC98-348.sub.BVDV vaccine primed BVDV-specific immune responses in the presence of neutralizing antibodies but failed to fully expand the primed responses. Failure to detect significant BVDV-specific immune responses and/or protection in group A as compared to all the other groups (B-E) after challenge will result in the rejection of the hypothesis.
b) Immunization of Neonatal Calves
(84) In one embodiment, the present invention contemplates testing whether immunization of neonatal calves with a single dose of a vaccine formulation containing adenoviruses expressing Flt3L/GM-CSF/CD40L for DC recruitment and activation, and DC-targeted hydrophilic domains of immunogenic BVDV antigens will prime and expand significant BVDV-specific B and T cell responses in the presence of neutralizing antibodies and confer protection upon challenge. Adenoviruses expressing the Flt3L/GM-CSF/CD40L cytokines (
(85) A single dose of the adenovirus-vectored BVDV vaccine should express sufficient amounts of the DC-targeted hydrophilic BVDV antigen chimeras (
(86) If the adenovirus-vectored cytokines fail to significantly enhance priming of BVDV-specific immune responses, an alternative strategy for augmenting the immunogenicity of the adenovirus-vectored BVDV vaccine is to determine whether replacing the AdCD40L with an adenovirus-vectored CD40 agonistic diabody generated from an anti-bovine CD40 agonistic mAb will improve vaccine immunogenicity (75). Agonistic anti-bovine CD40 mAbs have been generated and are currently being characterized. A second alternative strategy for augmenting BVDV vaccine immunogenicity is to use TLR 3 ligand (poly I:C), TLR 7/8 ligands (imiquimod and resiquimod), and TLR 9 (CpG ODNs) to improve vaccine efficacy (101).
(87) The hypothesis that intradermal inoculation of neonatal calves with a single dose of a vaccine formulation containing adenoviruses expressing Flt3L/GM-CSF/CD40L and DC-targeted BVDV antigens will prime and expand significant BVDV-specific B and T cell responses in the presence of neutralizing antibodies and confer protection upon challenge will be accepted if, after one immunization, a significant BVDV-specific immune responses and/or protection is detected in group A but none in group B (
(88) In one embodiment, the present invention contemplates two alternative strategies for further improving the proposed BVDV vaccine: 1) addition of defined DR-restricted T cell epitopes (QGGISSVDHVTAGKDLLV (SEQ ID NO: 6), VTASGTPAFFD LKNLKGW (SEQ ID NO: 7), VEYSYIFLDEYHCATPEQ (SEQ ID NO: 8), CATPEQLAIIGKIHRFSE (SEQ ID NO: 9), MKGNMLVFVPTRNMAVEV (SEQ ID NO: 10), GRVKPGRYYRSQETA (SEQ ID NO: 11), NGEVTDTYENYSFLNARK (SEQ ID NO: 12)) (28) from the conserved regions of the E2 and NS2-3 antigens from BVDV 1a and variants of these epitopes from the other BVDV genotypes to the BVDV antigen chimeras to generate a synthetic chimeric gene encoding a BVDV polyepitope; and 2) use short-term T cell lines from BVDV vaccinates and or convalescent animals to identify CTL epitopes from the pool of potential MHC class I allele-specific cytotoxic T cell peptide motifs from the BVDV polyprotein and reactive epitopes will be added to the BVDV polyepitope to generate a CD4.sup.+ and CD8.sup.+ epitope-rich vaccine candidate (28, 31).