Chimeric VLPs Bearing VP60 from Two Serotypes of Rabbit Haemorrhagic Disease Virus Are Protective against Both Viruses.

The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations.

A novel baculovirus-derived promoter with high activity in the baculovirus expression system.

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Production of functional active human growth factors in insects used as living biofactories.

Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications.

Significant productivity improvement of the baculovirus expression vector system by engineering a novel expression cassette.

Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10. The prolonged cell integrity observed in cells infected by baculoviruses harbouring the novel expression cassette reduced the characteristic proteolysis and aberrant forms frequently found in baculovirus-derived recombinant proteins. The new expression cassette developed here has the potential to significantly improve the productivity of the BEVS.

Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector.

The promoter sequences of the encoding genes for the three most abundant hexamerins of the Lepidoptera Trichoplusia ni were isolated and cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-derived baculovirus expression vector. From the sequences analyzed, the DNA region driving the expression of the Basic juvenile hormone-suppressible protein 2 (BJHSP-2), denominated pB2, presented the highest promoter strength in the context of the baculovirus vector in Sf21 insect cells. This promoter activity occurred earlier in baculovirus-infected cells than that achieved by a conventional polyhedrin promoter (polh), but surprisingly stopped at 48h post-infection. A mapping of pB2 essential promoter elements determined that a region of about 400bp, denominated pB29, retained and even increased the transcriptional activity with respect to the parental full-length sequence. Finally, several chimeric combinations of the insect-derived pB2 with the virus-derived conventional polh or p10 promoters were constructed and incorporated into an AcMNPV baculovirus vector. The pB2-p10 combination showed increased recombinant protein expression at early times post-infection and similar expression levels at very late times post-infection in Sf21 cells with respect to conventional late promoters. To the best of our knowledge, pB2 is the first promoter isolated from the Lepidoptera T. ni, the natural host of AcMNPV, to be assayed in a baculovirus expression vector.

African swine fever virus serodiagnosis: a general review with a focus on the analyses of African serum samples.

African swine fever (ASF) is an infectious disease that causes heavy mortality in domestic pigs. At present there is no vaccine against ASF, and eradication in countries where the disease is endemic is based only on competent diagnosis programs and the sacrifice of infected animals. Due to the presence of natural attenuated strains, certain infection conditions may result in reduced mortality. In these situations, the disease can be diagnosed by detection of specific antibodies. The use of classical and validated diagnosis assays, such as ELISA and Indirect Immunofluorescence or Immunoblotting, allowed the eradication of ASF in the Iberian Peninsula in the 1990s. However, given that conventional tests include the use of antigens obtained from ASF virus (ASFV)-infected cells, they have several disadvantages, such as difficulties to achieve standardization and also the risks associated with the manipulation of live virus. Such drawbacks have led to the development of alternative and more robust systems for the production of ASFV antigens for use in anti-ASFV antibody detection systems. In the present review, we provide an update on current knowledge about antigen targets for ASFV serodiagnosis, the significant progress made in recombinant antigen production, and the refinement of ASF serological diagnostic assays. Moreover, we describe the accuracy of an ELISA developed for the serodiagnosis of ASFV in Africa. This assay is based on a novel p30 recombinant protein (p30r) obtained from an Eastern African viral isolate (Morara strain), which shares 100% amino acid sequence identity with the Georgia virus isolate. That study included the analyses of 587 field sera collected from domestic pigs and warthogs in Senegal (West Africa), the Democratic Republic of Congo (Central Africa), Mozambique (South-East Africa), and South Africa. The results revealed that the novel p30r-based ELISA allows the accurate detection of antibodies against ASFV, independently of the geographical origin of the sera.

Head-to-head comparison of three vaccination strategies based on DNA and raw insect-derived recombinant proteins against Leishmania.

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines- is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo.

BACKGROUND: Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. RESULTS: Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. CONCLUSIONS: Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule.

Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae.

BACKGROUND: There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in E. coli, yeast and baculovirus grown in insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV). FINDINGS: Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in insect larvae. Recognition of G21-465 by ß-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (Oncorhynchus mykiss) was demonstrated. CONCLUSIONS: Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.

Humoral and in vivo cellular immunity against the raw insect-derived recombinant Leishmania infantum antigens KMPII, TRYP, LACK, and papLe22 in dogs from an endemic area.

Leishmania infantum causes visceral leishmaniasis, a severe zoonotic and systemic disease that is fatal if left untreated. Identification of the antigens involved in Leishmania-specific protective immune response is a research priority for the development of effective control measures. For this purpose, we evaluated, in 27 dogs from an enzootic zone, specific humoral and cellular immune response by delayed-type hypersensitivity (DTH) skin test both against total L. infantum antigen and the raw Trichoplusia ni insect-derived kinetoplastid membrane protein-11 (rKMPII), tryparedoxin peroxidase (rTRYP), Leishmania homologue of receptors for activated C kinase (rLACK), and 22-kDa potentially aggravating protein of Leishmania (rpapLe22) antigens from this parasite. rTRYP induced the highest number of positive DTH responses (55% of leishmanin skin test [LST]-positive dogs), showing that TRYP antigen is an important T cell immunogen, and it could be a promising vaccine candidate against this disease. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 82% of LST-positive dogs were detected, suggesting that both antigens could be considered as components of a standardized DTH immunodiagnostic tool for dogs.

Dynamics and predictive potential of antibodies against insect-derived recombinant Leishmania infantum proteins during chemotherapy of naturally infected dogs.

A predictive marker for the success treatment of canine leishmaniasis is required for the application of a more rational therapy protocol, which must improve the probability of cure and reduce Leishmania resistance to drugs. We investigated the dynamics and predictive value of antibodies against insect-derived recombinant L. infantum proteins rKMPII and rTRYP by using an enzyme-linked immunosorbent assay with retrospective serum samples from 36 dogs during treatment of canine leishmaniasis. In the entire group of dogs, concentrations of antibodies against rKMPII and rTRYP significantly decreased earlier than concentrations of antibodies against crude total Leishmania antigen (one versus six months), which suggested that the dynamics of antibodies against recombinant proteins may be useful for assessing clinical improvement after treatment. Interestingly, decreases in antibody concentrations against rKMPII occurred earlier in disease-free dogs than in dogs that remain clinically ill one year after beginning of treatment, which suggested that these antibodies may be useful for predicting disease-free survival one year after the beginning of therapy against canine leishmaniasis.

Immunity conferred by an experimental vaccine based on the recombinant PCV2 Cap protein expressed in Trichoplusia ni-larvae.

Porcine circovirus type 2 (PCV2) vaccination has been recently included as a measure to control postweaning multisystemic wasting syndrome (PMWS) in the field. Aiming to obtain a more affordable vaccine to be extensively implemented in the field, a highly efficient non-fermentative expression platform based on Trichoplusia ni (T. ni) larvae was used to produce a baculovirus-derived recombinant PCV2 Cap protein (rCap) for vaccine purposes. Vaccination of pigs with rCap induced solid protection against PCV2 experimental infection, inhibiting both the viremia and the viral shedding very efficiently. The protection afforded by the rCap vaccine strongly correlated with the induction of specific humoral immune responses, even in the presence of PCV2-specific maternal immunity, although cellular responses also seemed to play a partial role. In summary, we have shown that rCap expressed in T. ni larvae could be a cost-effective PCV2 vaccine candidate to be tested under field conditions.

DnaK/DnaJ-assisted recombinant protein production in Trichoplusia ni larvae.

The DnaK/DnaJ Escherichia coli chaperone pair, co-produced along with recombinant proteins, has been widely used to assist protein folding in bacterial cells, although with poor consensus about the ultimate effect on protein quality and its general applicability. Here, we have evaluated for the first time these bacterial proteins as folding modulators in a highly promising recombinant protein platform based on insect larvae. Intriguingly, the bacterial chaperones enhanced the solubility of a reporter, misfolding-prone GFP, doubling the yield of recombinant protein that can be recovered from the larvae extracts in a production process. This occurs without negative effects on the yield of total protein (extractable plus insoluble), indicative of a proteolytic stability of the chaperone substrate. It is in contrast with what has been observed in bacteria for the same reporter protein, which is dramatically degraded in a DnaK-dependent manner. The reported data are discussed in the context of the biotechnological potential and applicability of prokaryotic chaperones in complex, eukaryotic factories for recombinant protein production.

Seroreactivity against raw insect-derived recombinant KMPII, TRYP, and LACK Leishmania infantum proteins in infected dogs.

The recombinant proteins KMPII, TRYP, and LACK of Leishmania infantum were produced in baculovirus-infected Trichoplusia ni larvae and used to analyze the seroreactivity of 165 dog serum samples by the multiple-well ELISA technique (57 infected dogs with clinical signs, 46 naturally infected and 11 experimentally infected; and 108 non-infected dogs, 76 from non-endemic areas and 32 from endemic areas). Recombinant (r) KMPII was the most recognized antigen, as the majority of infected dogs seroreacted against it (0.75). This is the first report of seroreactivity against rTRYP (0.51) and rLACK (0.42) in L. infantum-infected dogs, since previous studies using recombinant TRYP and LACK proteins produced in prokaryotic systems failed to detect specific seroreactivity. All non-infected dogs were negative for rTRYP and rLACK, and only one of the 32 from endemic areas seroreacted against rKMPII. The results demonstrate that L. infantum-infected dogs develop humoral immunity against rKMPII, rTRYP, and rLACK antigens. There was substantial agreement between crude total L. infantum antigen (CTLA)-based ELISA and rKMPII ELISA (kappa=0.664), although this was higher than that found between the CTLA-based ELISA and rTRYP (kappa=0.427) or rLACK (kappa=0.343) ELISA, which can be interpreted as fair and moderate agreement, respectively. Ninety-three percent of the infected dogs analyzed developed specific antibodies against at least one of these three recombinant antigens. When the three recombinant antigen-based ELISA techniques were evaluated in parallel, almost perfect agreement (kappa=0.880) with CTLA-based ELISA was observed, with a specificity of 0.97 and a sensitivity of 0.93 in relation to CTLA-based ELISA. Further studies using purified recombinant antigens in a single-well test or individually, depending on the objective of the study, are warranted.

Development of two Trichoplusia ni larvae-derived ELISAs for the detection of antibodies against replicase and capsid proteins of porcine circovirus type 2 in domestic pigs.

The main aim of the present study was to describe new methods for the identification of antibodies against the PCV2 capsid (Cap) and replicase (Rep) proteins in pig sera. Specifically, two new indirect enzyme-linked immunosorbent assays (ELISA) were developed based on recombinant PCV2 Cap (rCap) and Rep/Rep' (rRep) proteins expressed in baculovirus and produced in Trichoplusia ni insect larvae. Both assays were validated by testing serum samples in a longitudinal study of 107 animals with different clinico-pathological features of PCV2 infection: pigs with postweaning multisystemic wasting syndrome (PMWS), wasted pigs without a diagnosis of PMWS and healthy animals. Longitudinal antibody profiles indicated that healthy animals had significantly higher anti-Cap and anti-Rep antibody levels than the rest of the animal groups at 11 weeks of age. Moreover, PMWS affected pigs could be distinguished from the rest of the pig groups by their lower anti-Rep antibody levels at 11 weeks of age and at necropsy. The results demonstrate the potential of these two ELISAs for large-scale serological studies. This study represents the first longitudinal study of the induction of anti-Cap and anti-Rep antibodies in farms affected by PMWS, from 1 week of age until the occurrence of disease.