DISCOVER ALGENEX.

Immunopotentiating molecule APCH1

A new molecule designed for targeting vaccine antigens to immune-competent cells, increasing vaccine potency and facilitating broad immune responses

2010 Alternative Gene Expression S.L.

BACKGROUND

Recombinant subunit and peptidic vaccine in general present a reduced immunogenicity in vaccinated individuals with respect to the whole pathogen from which they derived. The generation of strong immune responses to these vaccines requires the use of potent adjuvants, high antigen doses and repetitive vaccinations. The antibody response to most protein antigens requires specific cooperation between B and T cells. In order to deliver the helper signal, CD4+ T cells must recognize processed antigens in the context of the major histocompatibility complex Class II molecules (MHCII) found on the membrane of antigen presenting cells (APCs).

Vaccine antigens may be targeted to APCs by conjugating them to antibodies directed against surface molecules on APCs. This methodology was originally developed to enhance immune responses with low doses of antigen and without the use of adjuvants. It was also described to bias immune responses towards specific directions such as antibody production, CD4+ T-cell stimulation or even cytotoxicity-mediated immunity. In particular, MHC II-targeted antigens were described to enhance antigen presentation, producing strong primary humoral responses, both systemic and mucosal and generating long-term memory responses . This approach was reported to generate potent recombinant subunit vaccines that could be produced in adjuvant-free formulations.

Antigen

Dendritic cell

MODE OF ACTION
Targeting antibody

T cell
Vaccine antigen

Immunization
Class II antigens

Internalization

APCs
Antigen presentation by Class I and II molecules

Humoral and cellular immune responses

Mechanisms of intracellular trafficking previously described for B lymphocytes could be used to explain how MHC II targeted proteins and peptides may induce an enhanced antigen presentation. According to this, newly synthesized class II molecules may be targeted to endosomal compartments following transport to the cell surface and rapid internalization. Support for this hypothesis was obtained in studies showing that the N-terminal endosomal targeting signal in the cytoplasmic domain of class II-associated invariant chains (Ii) also functions as an efficient internalization signal, resulting in rapid endocytosis of MHC II-Ii complexes from the cell surface into endosomal compartments in which Ii dissociates from the complex. Once there, they get in contact with processed antigens and eventually they may bind peptides that will be later exposed on the cell membrane. In this way, MHC II-targeted antigens may follow this endocytic route to reach intracellular compartments where they can be processed and associated to other MHC II molecules for antigen presentation.

Hybridoma 1F12

APCH1 GENERATION
ALGENEX has developed a molecule (single chain antibody) for targeting vaccine antigens to APCs that increase subunit vaccines potency. For such purpose, we selected the monoclonal antibody 1F12 because it recognizes the chain of MHC II DR of different animal species including humans with high affinity. A recombinant single chain version of this antibody was obtained from the hybridoma cells to be used for obtaining transcriptional fusions with vaccine antigens. This single chain molecule was denominated APCH1.

Extraction of total RNA

Primers

RT-PCR

Amplification of cDNAs of the VH and VL domains from Mab 1F12

Primers

PCR

5 VH
3 VL

Annealing

PCR

APCH1 (ScFv)

Cloning strategy of APCH1 single chain antibody from hybridoma 1F12

Macrophage

Macrophage decorated with chimeric vaccines

Vaccine antigen
APCH1

Control- mAb APCH1-fusion

1F12 mAb
3

A
B

The functionallity of APCH1 fusions have been tested in vitro by analysis of the binding capacity to MHC II antigens present on the surface of APCs obtained from different animal species.

Binding of fusion protein APCH1-2L21 to alveolar pig macrophages in comparison to mAb 1F12 from which derived the single chain antibody APCH1. Surface decorated macrophages were visualized by immunohistochemistry (A) and immunofluorescence (B).

APCH1 ADVANTAGES:
APCH1 INCREASES ANTIBODY TITERS IN VACCINATED ANIMALS APCH1 molecule has been tested in a number of experimental vaccines in soluble form with excellent results.
1/serum dilution (log10)
GRUPO

Mice
1/serum dilution (log10)

Rabbits

4 3 2 1

4 3 2 1

Antibody response to peptide 2L21, recombinant APCH1-2L21, VP60 and APCH1VP60 in mice intraperitoneally immunized. Two immunization doses (5g) in presence of oil adjuvant.

Antibody immune responses obtained in rabbits immunized intramuscularly with a subimmunogenic dose of 10g of VP60 protein or with an identical dose of recombinant APCH1-VP60 fusion protein. Animals received a single dose and were challenged with infective RHDV at 30 dpi. Serum samples were taken at 15, 30 and 45 dpi and analyzed by ELISA. Rabbits immunized with VP60 died after RHDV infection, while rabbits immunized with the same dose of APCH1-VP60 survived to the virus infection and presented none or low increase in antibody titers measured 15 days after challenge exposure.

APCH1 REDUCES THE ANTIGEN REQUIRED IN VACCINES

Cows
6

1/serum dilution (Log10)

T0
3

T15

T30
2
T35

T55
1

408 161 422 194 452 78 182 403 421 151 460 163 444 75 459 190 82 197 384 408 445 161 167 422 119 194 175 452 397 78 105 182 92 403 387 421 174 151 396 460 461 163 458 444 449 459 93 82 393 384 170 445 154 167 453 119 171 175 389 397 94 105

92 387 174 396 461 458 449 93 393 170 154 453 171 389 94

The potentiating effect of APCH1 molecule has been tested in cows with an experimental vaccine against Bovine virus GRUPO diarrhea. BVD seronegative cows were immunized with T0 different vaccine formulations. Groups of 8 cows received the T15 following immunogens: T30 A: APCH1-E2: 0,5 gT35 /dose B: Purified E2: 2 g /dose T55 C: Crude extract containing E2: 10 g/dose Animals received 2 doses of vaccine at days 0 and 30 and were challenged with virus at day 60. Animals were monitored during 30 days

APCH1 CAN BE USED IN DNA VACCINATION AND INDUCES CELLULAR IMMUNITY

pCMV-APCH1BTT pCMV-APCH1BTT
#1
3,2%

pCMV
#1
23,2%

pCMV-APCH1BTT pCMV-APCH1BTT pCMV

#1 #2
0,22%
23,2% 0,09% 0,09% 8,7% 8,7% 0,02%

#2 #1
3,2% 0,02%

#3 #2
0,02% 2,54%

#4 #3
2,54% 0,03%

#4
0,22% 0,03%

pCMV
#4
0,06% 0,02%

pCM

#2 #3

#3 #4

IFNg

IFNg

CD4+

CD4+

IFNg

IFNg

CD8+

CD8+

pCMV-APCH1BTT induces specific T-cell responses in mice. Splenocytes from mice immunized with pCMV-APCH1BTT or with pCMV BTT were in vitro stimulated with a mixture of the three specific Foot and mouth disease virus (FMDV) synthetic peptides (1mg/ml of each one), corresponding to those encoded in the DNA vaccine (B, T3A and TVP4). Values correspond to the percentage of peptide-specific CD4+ or CD8+ T-cells expressing IFN gamma (after subtracting the values obtained in cells incubated with medium alone).
medium

BEI-inactivated FMDV

a
IFNg- secreting cells/ 106 PBMCs
35 30 25 20 15 10 5 0 1 2 3 4 5 6 0 1 2 3 4 5 80 60 40 20 100

Detection of IFNg by ELISPOT in DNAimmunized pigs. Average number of IFNgamma producing cells per 106 peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation with: 105 pfu/ml of BEIinactivated FMDV Cs8c1 (black bars) or medium alone (white bars) are shown. Standard deviation bars correspond to three replicates. Panel a: prior to viral challenge; panel b: at day 10 after viral challenge.
6

pCMV-APCH1BTT

pCMV

pCMV-APCH1BTT

pCMV

APCH1 FACILITATES SECONDARY IMMUNE RESPONSES AFTER VIRUS INFECTION IN VACCINATED ANIMALS
a
5 4
pCMV-APCH1BTT

c
#1 #2 #3 #4

3
pCMV

#5 #6

2 1 0
0 3 6 8 10 0 3 6 8 10 0 3 6 8 10

Days post challenge

Kinetics of seroneutralization in DNA-immunized pigs after viral challenge. Results are represented as PRN50, i.e., dilution of serum (log10) causing a reduction of 50% in the number of PFU in a plaque-reduction assay (21). Samples assayed were collected at day 43 post immunization (corresponding to day 0 relative to viral challenge), and at days 3, 6, 8 and 10 post challenge. Viral reductions are represented relative to the viral titers obtained with preimmune sera (collected at day 0 of the experiment, prior to DNA immunization). The viruses used in the assays were: FMDV C-S8 (panel a), FMDV C3Arg (panel b) and FMDV O1 Campos (panel c). Results are the mean of at least two independent experiments.

PRN50

Literature reference: -Gil et al.. Targeting antigens to an invariant epitope of the MHC Class II DR molecule potentiates the immune response to subunit vaccines Virus Research 2010 (in press) -Borrego et al.. Targeting FMDV minigenes to SLAII positive cells enhances the induction of cellular responses in swine and confers protection against viral challenge. The Global control of FMDV-Tools and Ideas- Erice, Italy 14-17. 2008