Large-scale production in Pichia pastoris of the recombinant vaccine Gavac™ against cattle tick

Elsevier

Vaccine, Vol. 15, No. 4, pp. 414-422, 1997 Copyright 0 1997 Elsevier Science Ltd. All rights resewed

Printed in Great Britain

ELSEVIER PII: S026440X(96)00192-2 0264-41ow97 $17+0.00

Large-scale production in Pichia pastoris of the recombinant vaccine Gavac@ against cattle tick

Mario Canales*, Antonio Enriquez *, Eduardo Ramos*, Deborah Cabrera*, Hubert Dandie”, Alejandro Soto *, Viviana Falcbn?, Manuel Rodriguezf and JosC de la Fuentet

A gene coding for the Bm86 tick protein was recently cloned, expressed in Pichia pastoris and shown to induce an inmunological response in cattle against ticks. Moreover, the Gavac@ vaccine (Heber Biotec S.A., Havana, Cuba), which contains this recombinant protein, has proved to control the Boophilus microplus populations underfield conditions. This paper reviews the development and large-scale production of this vaccine, the ejicacy of the resulting product and the strategy followed in designing its production plant, The production plant fulfills biosafety requirements and GMP. 0 1997 Elsevier Science Ltd.

Keywords: Boophilus microplus; vaccine; rBm86 protein; Gavac

The ectoparasite Boophilus microplus is a major veteri- nary problem as a vector of severe diseases and as a debilitating agent to cattle in tropical and subtropical climates. Its traditional control methods include the use of chemicals which, having partially successful results, are costly and cause environmental and residual problems. Hence, combining natural resistance with resistance induced in the hosts by vaccination is a new posibility for protecting cattle against tick infestations.

Partial protection with vaccines containing the recombinant Bm86 protein isolated from the plasmatic membrane of tick-gut epithelial cells and expressed on transformed cells of either Escherichia coli or Aspergillus niduians has been reported . ‘J Following this trend and using the rBm86 protein expressed in the yeast Pichia pastoris, we have developed a technology to produce Gavac@, a recombinant vaccine against B. microplus, whose effective control of tick infestations has been recently reported3-’ ‘.

The methylotrophic yeast P. pastoris was chosen as host for the cloning and expression of this protein because it has proved to be a suitable host in the industrial production of heterologous proteins12s’3X’4, requiring less expensive media and offering other advan- tages in terms of productivity, capital investment and operating costs compared to other host systems. The amount of rBm86 secreted in P. pastoris has been reported to be about 1.5-2.0 g l- m fermentations

*Division of Technological Development, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 6, Cuba; tAnalytical Unit, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 6, Cuba; IMamma- lian Cell Genetics, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 6, Cuba. (Received 16 May 1996; revised 7 August 1996; accepted 7 August 1996)

414 Vaccine 1997 .Volume 15 Number 4

using methanol as a sole carbon source, which is higher than those obtained in E. coli and A. nidulans’.

All biotechnological processes for vaccine production can be accommodated in six steps: cell multiplication; phase separation; product isolation; purification; polish- ing; and adjuvation and filling. Designing a process involves the definition of the necessary steps on the basis of all the information available, the properties of both the tial product and starting material, and the choice of the adequate equipment. In addition, a careful selection of the separation sequence to be employed after cell multiplication must be made, for the purpose of main- taining a high yield and obtaining a virtually pure product at reasonable cost15-18.

This article presents details on various design and production aspects of the recombinant DNA technology used to obtain the Gavac@ vaccine. The process developed was simple and cost-effective for industrial production, and allowed obtaining the glycosylated and particulated protein which we used as antigen in the vaccine formulation. The steps followed to design the plant for commercial purposes and the efficacy of the vaccine in the control of B. microplus infestations are also examined.

MATERIAL AND METHODS

Culture medimn preparation

A supplemented saline medium (SSM) containing ammonium sulfate, di-ammonium hydrogen orthophosphate, di-potassium hydrogen orthophosphate, calcium chloride, vitamins, traces of elements and 2% of glucose was used to cultivate the cells before seeding. Another medium with the same salts, yeast extract and vitamins was employed in the production fermentor. An initial concentration of 3.5% of glycerol and a flow of

Production of the recombinant vaccine Gavac”: M. Canales et al.

methanol of 2 ml l- ’ h- ’ were used during the growth and production phases.

Preparation of the master seed hank (MSB) The MSB was obtained from the P. pastoris strain

MB9, which contains a chromosomal copy of the gene coding for the Bm86 protein’. The strain was streaked in Petri dishes containing 15 ml of SSM and grown for 48 h at 30°C. An isolated colony was used to inoculate a 5 ml culture of SSM in a 50-ml erlenmeyer flask, which was then grown on an orbital shaker at 240 revs min-’ for 24 h at 30°C. The content of this preculture was used entirely tb inoculate a 50-ml SSM culture in a 500-ml erlenmeyer flask, and rowth resumed on an orbital shaker at 240 revs min- ? for 24 h at 30°C. After this, the content of the culture was mixed with 50 ml of sterile SSM+60% glycerol, distributed in l-ml aliquots in sterile, hermetic vials and stored at - 70°C.

Preparation of the working cell bank (WCB) A frozen aliquot of the MSB was used to streak a

Petri dish containing 15 ml of SSM. All the above- mentioned steps to prepare the MSB were repeated to prepare the WCB, and the vials were also stored at - 70°C.

Inocuhun preparation Four 250-ml erlenmeyer flasks inoculated with cul-

tures grown for 72 h in the SSM, were grown on an orbital shaker at 30°C for 24 h. Then a seed fermentor was inoculated with this culture. P. pastoris was grown in a batch culture at 30°C during 24 h.

Fermentation The production fermentor was operated in a fed-batch

mode. Cells were initially cultivated in a medium with glycerol. Upon depletion of glycerol, methanol was added to the culture to induce the production of the rBm86 protein. At the end, the fermented broth was cooled to lO”C, to minimize cell lysis.

Ceil harvesting The chilled fermented broth was centrifuged in order

to wash, concentrate and recover the cells. The concentrated cells were resuspended in two volumes of soft- water and then separated in a self-cleaning disc ty e centrifuge at 106OOg. This process was repeated twice. P, .

Cell disruption After cell harvesting, the cells were disrupted in a ball

mill disruptor, in continuous flow, using beads of 0.5- 0.75 mm dia, with a packing density of 85%. The concentrated cells were resuspended at 100 g l- ’ (dry wt) in phosphate buffer, before cell rupture”.

Pellet-cell washing The homogenate was pumped to a holding tank and

resuspended in buffer containing basically the same salts as those used in cell disruption. The same self-cleaning disc type centrifuge that was used for cell harvesting

was used again. The heavy phase was collected and resuspended in phosphate buffer with urea. After homogenization, the suspension was separated again by centrifugation. The process was then repeated, and, at the end, the slurry was resuspended in phosphate buffer and centrifuged in high-speed, tube-type centrifuges at 14ooogY

Denaturation-solubilization of the rBm86 protein The washed cell-debris particles were resuspended and

strongly homogenized in a buffer containing 8 M urea. After homogenization, the solution was slowly shaked in a holding tank and then a separation step was performed in tube-type centrifuges at 14000g19.

Renaturation and concentration of rBm86 The supematant of solubilization containing the

rBm86 protein was homogenized and slowly diluted by addition of EDTA, and Na,HPO, and NaOH buffer. A concentration step was then performed by ultrafiltration”.

Precipitation of contaminant proteins with ammonium sulfate

Ammonium sulfate was added to the protein solution and homogenized at room temperature, after which a separation step was carried out by centrifugation in a tube-type centrifuge at 14000g’9.

Concentrationldiafiltration The supematant was concentrated and diafiltered in

two membrane processes using a buffer containing Na,HPO,, and NaH,P0,‘9.

Sterilizing filtration The protein solution was subjected to a sterilizing

filtration using a flat-plate filter, mounted in an stainless steel filter holder. Two passes through this system were performed to obtain a sterile antigen solution”.

Adjuvation of oily vaccine A solution of anhydromannitoletheroctodecenoate

(Montanide 888) in mineral oil, and the rBm86 protein solution were mixed in order to obtain a water-in-oil (w/o) emulsion, in a batch-by-batch process using an In-line Ultraturrax high-speed mixer”.

Fiing The vaccine was filled in a dosing machine in plastic

(high density polyethylene) bottles of 30, 50, and 100 ml.

Microbial purity test This test is described in the USP XXII. It was

performed in the MSB, the WCB, and in the inoculum before seeding.

Sterility test The sterility test for active raw material (ARM) was

performed according to WHO standards2’. For sterility

Vaccine 1997 Volume 15 Number 4 415


testing of bulk material, at least 5 ml were used for inoculation into each culture medium and for each incubation temperature.

Abnormal toxicity test The abnormal toxicity test was performed in mice

according to the principle described in the BP88.

Determination of protein concentration Protein concentrations were determined as described

by Lowry et al.”

Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE)

These were performed as described by Laemmli2’ in 10% gels with samples under standard reduction and denaturation conditions. Samples were prepared with a two times concentrated buffer sample. Ten microliters of rBm86 protein solution (12-15 ,ug of ARM) were applied when the Coomassie staining was used.

Laser scanning densitometry For Coomassie Blue stained gels, densitometry was

performed in a LASER Densitometer (LKB Bromma 2202 Ultroscan).

Direct electron microscopy (DEM) The method used for direct observation through

negative staininqj was the same as that described by Montesino et al.

Immunocytochemistry study The samples were fixed with 2% glutaraldehyde and

post-fixed for 2 h in 1% 0~0,. They were dehydrated in increasing ethanol concentrations. The embedding was as in SpurZ4. The blocks were sectioned with an ultra- microtome (LKB 2188) and the ultrathin sections were placed on 400 mesh grids and were incubated in 1% PBS-BSA buffer with 0.02 M glycine. Grids were then incubated with a conjugate of colloidal gold and anti-rBm86 mouse monoclonal antibody, and the sections were examined in a JOEL-JEM 2000 EX electron microscope.

Sandwich ELISA for rBm86 protein determination The procedure employed to determine rBm86 protein

concentration was described by Triguero et al.”

Determination of mechanical stability To determine the stability of the emulsion by gravita-

tional force effects, 10 ml of emulsion were poured into two 15-ml centrifuge tubes. The height of the emulsified column (H,,) was measured in the tubes and then they were centrifuged in a 30-cm radius centrifuge for 1 h at 3000 revs min- ‘. The emulsified column height was

416 Vaccine 1997 Volume 15 Number 4

measured again (H,). The ratio H,/I$, was calculated. (This relation must be equal to or higher than 0.8 to fulfill the quality parameter for the vaccine.)

Thermal stability

Ten milliliters of the emulsion were emptied into 15ml centrifuge tubes. The height of the emulsified column (H,) in the tube was measured. The tubes were placed in contact with a thermostatic bath at 37.0 &O.l”C for a 15-day period, after which only the emulsified column was measured again (H,). The H,IH, rate was then calculated. (This relation must be equal to or higher than 0.9 to fulfill the quality parameter for the vaccine.)

Immunogenic potency determination Several dilutions were prepared for testing the vac-

cine. Five groups of 5-week-old Balb/c female mice, each group of 10-l 5 animals, were used for each vaccine dose to be tested. A control group was immunized with the standard vaccine and a placebo group innoculated with emulsion without rBm86 protein. Mice were bled at 42 days and tested for anti-rBm86 antibodies”.

RESULTS AND DISCUSSION

Production process for the g

reparation of the recombinant vaccine Gavac against cattle tick

Figure I shows the step sequence followed to produce the vaccine applying the seed-batch principle. This means that a suitable quantity of the recombinant strain is maintained as a master seed lot stored in individual vials at - 70°C. This seed stock can be stored for many years and each fermentation run starts from the same original seed preparation. The MSB and the WCB prepared complied with the sterility test and lo6 cells ml-’ were obtained in the initial viability test. The plasmid integration, the stability, and conservation of the genetic information were confirmed by Southern blot analysis.

In the fermentation process, when the cell mass grew in the medium with glycerol, growth was rapid but heterologous gene expression was fully repressed. Upon depletion of glycerol, the addition of methanol to the culture induced rBm86 protein production with slightly more cell growth. The protein expressed was distributed in the membrane system of the cells (Figure 2). A cell density of 65 g l- ’ (dry wt) was achieved by the addition of methanol as a sole carbon source, plus vitamins and traces of elements during fermentation.

In cell harvesting, the broth was concentrated three times to a cell concentration of 140 g l- ’ (dry wt) in a self-cleaning disc type centrifuge, obtaining a cell recovery above 95%.

Five passes of the cell suspension obtained after cell harvesting through the ball mill were sufficient to achieve the complete disruption of the cells. The rBm86 protein remained associated with the cell debris (Figure 3A).

While washing the cell pellets, the three steps described made it possible to remove about 65% of the contaminant proteins, with a 70% recovery. The volume of material was reduced to almost 1115 after the

Production of the recombinant vaccine Gavac”“: M. Canales et al.

SECTION v MEDIA PREPARATION - INOCULUM PREPARATION

,&, I ,

buffer ----+*I PELLET-CELL WASHING - supernatant

I

buffer -_) DENATURATION-SOLUt3ILlZATlON - cells debris

i particles

buffer -(REFqLDlNGl

DOWNSTREAM * SECTION CONCENTRATION - waste

buffer -*I

buffer - ULTRAFILTRATION-DIAFILTPATION - waste

t

VACCINE FORMULATION

Montanide 888

a FILLING

GavacTM vaccine

Figure 1 Process block diagram to obtain the recombinant Bm86 antigen and to produce the vaccine

last separation process was performed in tube-type the salt concentration was increased to 25%. Then, with centrifuges. The rBm86 was never observed dis- an efficient separation process by centrifugation, a solved in the supernatants of the washed pellet cells particle-free supernatant containing the rBm86 protein (Figure 3B). was obtained.

When the washed cell-debris particles were resuspended in a buffer containing 8 M urea, denaturation and solubilization of the rBm86 occurred. From 80 to 90% of the rBm86 associated to the cell debris was released. After that, a separation step was performed in the same tube-type centrifuges that were used for the concentration step of pellet-cell washing.

After the separation process, renaturation served the purpose of refolding the rBm86 in order to regain its original conformation. The supernatant containing the rBm86 protein was homogenized and slowly diluted by the addition of Na2HP0, and NaOH buffer. In this condition, disulfide bridges were completely deoxidized and the native structure of rBm86 protein was recov- ered. A concentration step was then performed by ultrafiltration. The rBm86 obtained has a particulated structure, and particle sizes obtained were in the range of 1740 nm (Figure 4).

This solution was concentrated and diafiltered in two membrane processes. Protein concentration was increased by ultrafiltration. Then, adding five volumes of the adequate buffer in the diafiltration process, the initial salts of the solution were completely removed. At the end, the protein solution was subjected to a sterilizing filtration. Two passes through the filtration system were sufficient to obtain a sterile antigen solution, which constitutes the ARM of the vaccine. The ARM was a slightly yellowish liquid without any particle either pre- cipitated or in suspension, and a purity of over 50%. Table I shows the ARM controls which were made for each ARM batch and their acceptance limits.

A precipitation step, based on the difference of solu- bility between contaminant proteins and rBm86, made it possible to precipitate most of the contaminants when

To produce the vaccine, the rBm86 protein solution was mixed in a high-speed mixer, with an oily phase containing Montanide 888 and mineral oil. The emulsion obtained after mixing, complied with the Oswald model for non-Newtonian fluids with viscosity-index values (u) < 1500 mPa s _ ’ and flow-index values (n) between 0.5 and 0.9. The oily vaccine was then subjected to quality control tests (Table 1).


Production of the recombinant vaccine Gavac”“: M. Canaies et al.

Figure 2 Electron photomicrograph of a thin section of the recombinant strain MB9 of /? pastoris grown on methanol as the sole carbon source. The internal distribution of the Bm86 protein is marked with a monoclonal antibody conjugated with colloidal gold (black grains)

Plant design for the production of Gavac@ The production plant was designed and its capacity

defined taking into account the yields and recovery rates obtained in the development of the process and the results of the market research. The final fermentation volume was selected, the fermentation process was scaled up, and the batch size was then fixed. Depending on their characteristics, the subsequent operations were scaled up too.

Considering the volume of materials to be handled and the recovery at each step, the flow diagram of the process was performed. The size of the processing equipment was estimated by the fermentation dump frequency and the maximum residence time allowable for each product in each step. Both mass and energy balances were made. Also, in order to support the technological-equipment selection, the physico-chemical properties of the main-process streams were determined.

Besides the time required by each step, storage times for the materials used in the process were estimated. All this information allowed us to select the technological equipment. Other process parameters employed for this purpose included: batch and continuous operation; solid content type and its size (where appropriate); contaminant types; concentration and removal rate; product yield; and accessory equipment. Once the equipment was selected, its size was determined on the basis of parameters such as: product residence limitation; size of solids; loading and removal efficiency; volumetric throughput rate; and heat-transfer area.

The quantities required of raw materials and inputs, and the storage capacities for these and products in process were calculated. Also, the requirements of

43.0 kD

4:

25.0 kD

‘,Z>>“’ i( 18.4 kD 14.3 kD

.;$j;’

.-

6

1 2 3 4 5 6 7 8

rBm86 AOX 67.0 kD

43.0 kD 6

25.0 kD

Figure 3 SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue. (A) Supernatants of the 1-5 passes through the bead mill and the pellet of the last pass. Lines: 1, rBm86; 2, molecular weight markers: Bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), /I-lacto-globulin (18.4 kDa) and lisozyme (14.3 kDa); 3, suspension of cells; 4-8, supernatants of the passes through the mill; 9, pellet of the last pass. (B) Streams of the pellet-cell washing process. Lines: 1, rBm86; 2, molecular weight markers; 3, cell debris after disruption; 4-7, supernatant of each separation process; 8, pellet of the last separation

steam, air for fermentors and instrumentation, air con- ditioning, vacuum, and water were calculated in order to define and size the utility equipment needed. A reasonable excess capacity was taken into account in order to support future expansion of the plant.

The layout of the production plant was made. In its design, two considerations were kept in mind: the amount of space allocated to each process area and the arrangement of both the process and the auxiliary equipment. Adequate space was provided around and above each piece of equipment to allow for its operation and maintenance, and to accommodate the accessory equipment. Utility connections between units and auxiliary equipment were designed in a way that they interfered as little as possible with access to each one by operators and maintenance personnel. An area to re- ceive the new equipment, parts and raw materials was



“““- d ktribution (%)

T-----l

202428323

F&tide sizz (nn-@

Figure 4 Particle size distribution of the rBmf36 protein obtained after renaturation

Table 1 Raw material and vaccine controls and parameter acceptance limits

Type of control Acceptance limits Method

Active raw material Specific activity Purity Subunits aggregation Thiomersal content Sterility

50% Electrophoresis: 86 KD band, 50% Particle mean diameter16 nm 0.045~.055 g I-’ To comply

ELISA rBm86, Lowry SDS-PAGE (LKB laser densitometer) Electron microscopy Spectrophotometry WHO Series 1973, No. 530

Vaccine Organoleptic characteristics Volume Viscosity Particle size distribution Mechanical stability Thermal stability Sterility Abnormal toxicity in mice Potency in mice

White brilliant creamy liquid lO4i2 ml cl 500 mPa s-’ 80%<5 pm H,,lH&O.a 37°C for 15 days H,,/H,,>O.9 To comply To comply To comply

assigned elsewhere in the building. The raw material was stored first in “quarantine”, examined for acceptability (identity tests were run if necessary for compliance with current GMP) and then released for use. The utility equipment was separated from the rest of the plant in another building and the chemical and micro- biological control areas were also located in a separate laboratory.

The production plant designed had an adequate staff of well-trained personnel. The personnel needs were defined on the basis of the organization of the production process. The plant operated on a 24 h-a-day, 7 days-a-week working regime. We divided the 168 h of the week into seven shifts of 24 h. The lines of authority were clearly established and the staff was organized to ensure the continuous presence of operators, super- visors, engineers and technicians in the plant. In

Visual appearance Volumetric measuring Rheologic study Microscopic counting Centrifugation test Stability test USP XXII BP88 Potency test

addition, it was organized in such a way that all parts of the plant’s operation were adequately supervised by the managing staff.

For every routine activity of the plant staff, standard operation procedures were established in step-by-step instructions written in a way comprehensible to all. The quality control records were also prepared. These included analytical reports to show that each operation complies with the prescribed specifications described in the standard operating procedure. The decision regard- ing the release or rejection of the corresponding product in process or end product was also reflected in this record. Other records for each batch of ARM and each vaccine batch were prepared. These manufacturing records provided a complete account of the manufacturing history of each batch produced, reflecting all steps in the manufacturing process and testing.



Table 2 Effect of vaccination with Gavac”

Experiment Tick strain DT

% Reductiona

DO DF Efficacy (%)”

1 Camcord 74 60 2 Camcord 30 61 3 Camcord 46 43 4 Yeerongpilly 43 50 5 Cenapa 67 46 6 Tuxpan 9 38 7 Tuxpan 43 22 8 Mora 19 26 9 Colombian field 41 26 10 Brazilian field 40 8

;‘3 10 11 12 13 OC 3oc 8.5 10.2

91 76 72 75 84 51 56 58 60 51

aDT, adult female ticks; DO, egg laying capacity; DF, fertility. bEfficacy (o/)=100-[l-(CRTxCROxCRF)]; CRT, reduction in the number of adult female ticks; CRO, reduction in the egg laying capacity; CRF, reduction in fertility. ‘The assay of effect on fertility was not completed

Table 3 Effect of vaccination with Gavac- in the control of tick infestations in the field

Location Breed” Time (in weeks)= $ximal controlb /a References

Limonar (Matanzas, Cuba) Holstein 28-31 81 3 Fazenda Resgate Cross-bred (dairy) 36 79 10 (Sao Paulo, Brazil) Cross-bred (beef) 24 96 10

B. indicus 24 97 10 Corrientes (Argentina) Cross-bred (beef) 20 55 6

“Cross-bred cattle were B. taurusxB, indicus cattle. bMeasured by counting the number of adult female ticks on the animals. CTime after the first injection with Gavac-. “Measured with respect to a nonvaccinated control group

Cost-effective analyses were performed to determine the feasibility of the investment. Fixed and total capital investments were estimated taking into account the cost of the equipment. Manufacturing costs, including depre- ciation, and the return on investment were calculated. The lifetime of the project was set at 10 years, and cash flows for that period were obtained. Finally, supported by the cost-effective analysis, the investments required were made and the production process was established.

Efficacy of vaccination with Gavac@

The vaccine was registered in Cuba in 1993, in Colombia and the Dominican Republic in 1994 and in Brazil, Paraguay and Bolivia in 1995. Trials under controlled conditions have been conducted in Mexico26, Brazil’, Colombia8, and Cuba’ and under field conditions in Cuba3.4, Brazil”, Argentina6, and Colombia’.

The vaccination scheme recommended includes two vaccinations four weeks apart, and a third vaccination 3 weeks later3.7. Antibodies to the rBm86 protein are raised in the cattle during vaccination. Subsequent in- gestion by ticks of blood from vaccinated cattle leads to the binding of the antibody to this cell surface antigen and to the destruction of the ticks’ gut digestive cells2’. The damage appears in four ways: a reduction in the number of ticks engorging on vaccinated cattle; a reduction in the weight of the engorging females and the tick’s egg-la

Y ing capacity; and finally, in the viability of the

eggs . 3, 327 The major effect is thus not to kill the ticks currently infecting the herd of cattle but to reduce the posterior contamination, and thereby the parasite challenge in the next generation27.


The effect of the vaccine was determined employing several parameters, such as: the number of adult female ticks; the effect on the weight of the engorged ticks; the survival of adult female ticks; the egg-laying capacity; its fertility; and finally, the efficacy is determined taking into account the reduction in the number of adult female ticks, the egg-laying capacity and fertility’.

In ten experiments conducted to evaluate the efficacy of the Gavac@ vaccine against different B. microplus strains and on different cattle breeds, the efficacy was found to be ~50%’ (Table 2). These experiments were conducted under controlled conditions that did not exactly reflect the conditions found in the field, but were necessary for control studies. Nevertheless, in field experiments with Gavac@, it was demonstrated that the vaccine controls B. microplus populations in grazing cattle3*4,6,9.‘0.1 ’ (Table 3).

In Cuba, a combination of chemical acaricide treatments and vaccination was used in a schedule aimed to reduce the tick population in a cattle farm at the “Los Naranjos” enterprise. This combined method allowed a reduction of 50% of the chemical acaricides consumed and a very low tick number was obtained4 (Table 4).

Similar results were obtained using acaricide treatments and vaccination in Colombia’ and Brazil’ ’ (Table 4). The interval between acaricide treatments was ex- tended from 12 days to an average of 35 days with 60 days as maximum, depending on the cattle breed. This represented an average 57% reduction in the number of acaricide treatments in a year and savings of $3 per animal per year, plus a reduction in the problems associated with acaricide treatments’.

Other advantageous effects have been observed during vaccination with Gavac@. The incidence of Babesiosis

Production of the recombinant vaccine Gava?: M. Canales et al.

Table 4 Effect of vaccination with Gavac” on the frequency of acaricide treatments

Time between treatments (in days)b

Before After increase in time between

Location Breeda Vaccination treatments References

Los Naranjos (La Habana, Cuba) Holstein 7 22 15 4 Cross-bred 14 40 26 9 Holstein 15 27 12 9

Colombia B. in&us 15 75 60 9 Barra Mansa (Rio de Janeiro, Brazil) Holstein 12 27 15 11

%ross-bred cattle were B. taurusxB. in&us cattle. bRepresents the average values of several experiments including cattle with one to four injections with Gavac”

averaged 5.7 cases/1000 cattle before vaccination in “Los Naranjos” and, after the first injection, this ratio dropped to an average of 1.5 cases of Babesiosis/lOOO cattle in the following 10 months4. In Colombia, an effect of the vaccine in the reduction of Anaplasmosis was also recorded’, and, in Brazil, a dramatic reduction in the number of fly (Dermatobia spp. and Haematobia irritans) larvae on the animals was recorded”. Since B. microplus is a vector of Babesia bovis and Anaplasma sp., the effect of the vaccine on the transmission of these microorganisms was probably a result of vaccination against B. microplus infestations439 and, although it is very unlikely that the vaccine would have an effect on flies, the above-mentioned reduction probably reflected an improvement in the health of the animals after vaccination with Gavac@’ ‘.

CONCLUSION

Any attempt to reduce the incidence of tick infestations on cattle, inducing the artificial resistance of the host, necessitates the availability of large quantities of potent, safe, and affordable vaccines. The recombinant DNA technology developed by us allowed the obtainment of an effective vaccine which made this objective achiev- able. The scaling up and plant design for the production of the Gavac@ vaccine allowed the establishment of a stable production. The process designed was simple and cost-effective for industrial production, and proved to be an economical mean of making an efficient vaccine against ticks in potentially unlimited quantities. The vaccine obtained has shown efficacy in the reduction of B. microplus infestations.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the encouraging support and technical assistance of researchers and technicians from the CIGB involved in work with the vaccine, researchers and workers from the Estaci6n National de Parasitologia, the Board of Direction of the Instituto de Medicina Veterinaria, and researchers from Mexico, Colombia, Brazil, and Argentina. This work was supported by Hebert Biotec S.A. (Cuba), Consul- mar Emprendimentos Industriais e Comerciais Ltda. and Centrais Banana1 Agropecuaria Ltda. (Brazil), Minerales Exclusives y Cia. SCA. (Colombia), Revetmex S.A de C.V (Mexico) and Chemotecnica Sintyal S.A. (Argentina).

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Rand, K.N., Moore, T. and Sriskantha, A. et al. Cloning and expression of a protective antigen from the cattle tick Boophilus microplus. Proc. Nat/ Acad. Sci. USA 1989, 86, 9657-9661 Turnbull, I.F., Smith, D.R.J., Sharp, P.J., Cobon, G.S. and Hynes, M.J. Expression and secretion in Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal am& promoter system. Appl. Environ. Microbial. 1990, 56, 2847-2852 Rodriguez, M., Penichet, M.L. and Mouriz, A.E. et a/. Control of B. microplus populations in grazing cattle vaccinated with the recombinant Bm86 antigen preparation. Vet. Parasitol. 1995, 57,339-349 Rodriguez, M., Montero,C., Labarta, V. and de la Fuente, J. Effect of vaccination with GAVAC” on the incidence of Babesia bovis infections and the reduction in the number and frequency of acaricide treatments in cattle under production conditions in Cuba. In: Recombinant Vaccines for the Control of Cattle Tick (Ed. de la Fuente, J.). Elfos Scientiae, La Habana, Cuba, 1995, pp. 187-l 94 Masstird, C.L., da Fonseca, A.H., Pinheiro, V.R.E., de Oliveira, J.B. and Moraes da Silva, K.M. Evaluation of recombinant rBm86-“GAVAC” vaccine against the tick Boophilus microplus in Brazil. R. Bras. Med. Vet. 1995, 17, 167-173

Lambetti, J., Signorini, A., Mattes, C. et a/. Evaluation of the recombinant vaccine against Boophilus microplus in grazing cattle in Argentina. In: Recombinant Vaccines fir the C&intro/ if Cattle Tick (Ed. de la Fuente. J.). Elfos Scientiae. La Habana. Cuba, 1995: pp. 205-227 ’ Rodriguez, M., Rubiera, R. and Montesinos, R. et al. High level expression of the B. microplus Bm86 antigen in the yeast I? pastoris forming highly inmunogenic particles for cattle. J. Biotechnol. 1994, 33, 135-l 46

de la Fuente, J., Rodriguez, M., Fragoso, H. et a/. Efficacy of vaccination with Gavac- in the control of Boophilus microplus infestations. In: Recombinant Vaccines for the Control of Cattle Tick (Ed. de la Fuente, J.). Elfos Scientiae, La Habana, Cuba, 1995, pp. 177-185

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Large-scale production in Pichia pastoris of the recombinant vaccine Gavac™ against cattle tick

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