MITO END-3: efficacy of avelumab immunotherapy according to molecular profiling in first-line endometrial cancer therapy.

BACKGROUND: Immunotherapy combined with chemotherapy significantly improves progression-free survival (PFS) compared to first-line chemotherapy alone in advanced endometrial cancer (EC), with a much larger effect size in microsatellite instability-high (MSI-H) cases. New biomarkers might help to select patients who may have benefit among those with a microsatellite-stable (MSS) tumor. PATIENTS AND METHODS: In a pre-planned translational analysis of the MITO END-3 trial, we assessed the significance of genomic abnormalities in patients randomized to standard carboplatin/paclitaxel without or with avelumab. RESULTS: Out of 125 randomized patients, 109 had samples eligible for next-generation sequencing analysis, and 102 had MSI tested. According to The Cancer Genome Atlas (TCGA), there were 29 cases with MSI-H, 26 with MSS TP53 wild type (wt), 47 with MSS TP53 mutated (mut), and 1 case with POLE mutation. Four mutated genes were present in >30% of cases: TP53, PIK3CA, ARID1A, and PTEN. Eleven patients (10%) had a BRCA1/2 mutation (five in MSI-H and six in MSS). High tumor mutational burden (≥10 muts/Mb) was observed in all MSI-H patients, in 4 out of 47 MSS/TP53 mut, and no case in the MSS/TP53 wt category. The effect of avelumab on PFS significantly varied according to TCGA categories, being favorable in MSI-H and worst in MSS/TP53 mut (P interaction = 0.003); a similar non-significant trend was seen in survival analysis. ARID1A and PTEN also showed a statistically significant interaction with treatment effect, which was better in the presence of the mutation (ARID1A P interaction = 0.01; PTEN P interaction = 0.002). CONCLUSION: The MITO END-3 trial results suggest that TP53 mutation is associated with a poor effect of avelumab, while mutations of PTEN and ARID1A are related to a positive effect of the drug in patients with advanced EC.

Identification and initial characterization of matrix metalloproteinases in the yellow fever mosquito, Aedes aegypti.

Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.

Intrahaemocoelic infection of Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus does not induce tracheal cell basal lamina remodelling.

Infection of the lepidopteran insect Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) by the oral route stimulates activation of host matrix metalloproteases (MMP) and effector caspases, a process dependent on expression of the viral fibroblast growth factor (vFGF). This pathway leads to tracheal cell basal lamina remodelling, enabling virus escape from the primary site of infection, the midgut epithelium, and establishment of efficient systemic infection. In this study, we asked whether the MMP-caspase pathway was also activated following infection by intrahaemocoelic injection. We found that intrahaemocoelic infection did not lead to any observable tracheal cell or midgut epithelium basal lamina remodelling. MMP and caspase activities were not significantly stimulated. We conclude that the main role of the AcMNPV vFGF is in facilitating virus midgut escape.

The Autographa californica M nucleopolyhedrovirus ac79 gene encodes an early gene product with structural similarities to UvrC and intron-encoded endonucleases that is required for efficient budded virus production.

The Autographa californica M nucleopolyhedrovirus (AcMNPV) orf79 (ac79) gene is a conserved gene in baculoviruses and shares homology with genes in ascoviruses, iridoviruses, and several bacteria. Ac79 has a conserved motif and structural similarities to UvrC and intron-encoded endonucleases. Ac79 is produced at early times during infection and concentrates in the nucleus of infected cells at late times, suggesting a cellular compartment-specific function. To investigate its function, an ac79-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration assays showed that budded virus (BV) production was reduced in the ac79-knockout virus compared to control viruses, following either virus infection or the transfection of bacmid DNA. The ac79-knockout virus-infected cells produced plaques smaller than those infected with control ac79-carrying viruses. No obvious differences were observed in viral DNA synthesis, viral protein accumulation, or the formation of occlusion bodies in ac79-knockout and control viral DNA-transfected cells, indicating progression into the late and very late phases of viral infection. However, comparative analyses of the amounts of BV genomic DNA and structural proteins in a given quantity of infectious virions suggested that the ac79-knockout virus produced more noninfectious BV in infected cells than the control virus. The structure of the ac79-knockout BV determined by transmission electron microscopy appeared to be similar to that of the control virus, although aberrant capsid protein-containing tubular structures were observed in the nuclei of ac79-knockout virus-infected cells. Tubular structures were not observed for ac79 viruses with mutations in conserved endonuclease residues. These results indicate that Ac79 is required for efficient BV production.

Viral fibroblast growth factor, matrix metalloproteases, and caspases are associated with enhancing systemic infection by baculoviruses.

Most arthropod-borne and invertebrate viruses are orally ingested and commence infection in cells of the invertebrate intestine. Infection of secondary sites and eventual transmission to other hosts is hindered by basal lamina, a tightly interwoven and virus-impenetrable noncellular layer, lining the intestine and other organ cell layers. The mechanisms for viral escape across basal laminae are unknown. We describe an elegant mechanism mediated by a baculovirus-encoded fibroblast growth factor (vFGF) that signals a previously undescribed stepwise cascade of protease activation wherein matrix metalloproteases activate effector caspases, leading to remodeling of basal lamina lining tracheal cells associated with the intestine and culminating in the establishment of efficient systemic infections. Because FGFs coordinate diverse functions during development, metabolic processes, and tissue repair, it is plausible that the vFGF-mediated pathway described here is widely used during developmental and pathogenic processes that involve basal lamina remodeling.

Transcriptional Reprogramming of Autographa Californica Multiple Nucleopolyhedrovirus Chitinase and Cathepsin Genes Enhances Virulence.

The baculoviral chitinase (CHIA) and cathepsin (V-CATH) enzymes promote terminal insect host liquefaction, which aids viral progeny dissemination. Recombinant Autographa californica nucleopolyhedrovirus (AcMNPV)-derived viruses were previously generated with reprogrammed chiA transcription by replacing the native promoter with the AcMNPV polyhedrin (polh) or core protein (p6.9) promoter sequences, but of both these chiA-reprogrammed viruses lacked v-cath transcription and V-CATH enzymatic activity. Here, we report that dual p6.9/polh promoter reprogramming of the adjacent chiA/v-cath genes resulted in modulated temporal transcription of both genes without impacting infectious budded virus production. These promoter changes increased CHIA and V-CATH enzyme activities in infected Spodoptera frugiperda-derived cultured cells and Trichoplusia ni larvae. In addition, larvae infected with the dual reprogrammed virus had earlier mortalities and liquefaction. This recombinant baculovirus, lacking exogenous genomic elements and increased chiA/v-cath expression levels, may be desirable for and amenable to producing enhanced baculovirus-based biopesticides.

An Italian multicenter retrospective real-life analysis of patients with brain metastases from renal cell carcinoma: the BMRCC study.

BACKGROUND: The treatment of patients with brain-spread renal cell carcinoma (RCC) is an unmet clinical need, although more recent therapeutic strategies have significantly improved RCC patients' life expectancy. Our multicenter, retrospective, observational study investigated a real-world cohort of patients with brain metastases (BM) from RCC (BMRCC). PATIENTS AND METHODS: A total of 226 patients with histological diagnosis of RCC and radiological evidence of BM from 22 Italian institutions were enrolled. Univariate and multivariate models were performed to investigate the impact of clinicopathological features and multimodal treatments on both overall survival (OS) from the BM diagnosis and intracranial progression-free survival (iPFS). RESULTS: The median OS from the BM diagnosis was 18.8 months (interquartile range: 6.2-43 months). Multivariate analysis confirmed the following as positive independent prognostic factors: a Karnofsky Performance Status >70% [hazard ratio (HR) = 0.49, 95% confidence interval (CI) 0.26-0.92, P = 0.0026] and a single BM (HR = 0.51, 95% CI 0.31-0.86, P = 0. 0310); in contrast, the following were confirmed as worse prognosis factors: progressive extracranial disease (HR = 1.66, 95% CI 1.003-2.74, P = 0.00181) and only one line of systemic therapy after the BM occurrence (HR = 2.98, 95% CI 1.62-5.49, P = 0.029). Subgroup analyses showed no difference in iPFS according to the type of the first systemic treatment [immunotherapy (IT) or targeted therapy (TT)] carried out after the BM diagnosis (HR = 1.033, 95% CI 0.565-1.889, P = 0.16), and revealed that external radiation therapy (eRT) significantly prolonged iPFS when combined with IT (10.7 months, 95% CI 4.9-48 months, P = 0.0321) and not when combined with TT (9.01 months, 95% CI 2.7-21.2 months, P = 0.59). CONCLUSIONS: Our results suggest a potential additive effect in terms of iPFS for eRT combined with IT and encourage a more intensive multimodal therapeutic strategy in a multidisciplinary context to improve the survival of BMRCC patients.

Baculovirus infection induces a DNA damage response that is required for efficient viral replication.

Several mammalian viruses have been shown to induce a cellular DNA damage response during replication, and in some cases, this response is required for optimal virus replication. However, nothing is known about whether a DNA damage response is stimulated by DNA viruses in invertebrates. Cell cycle arrest and apoptosis are two of the downstream effects of the DNA damage response, and both are stimulated by baculovirus infection, suggesting a possible relationship between baculoviruses and the DNA damage response. In the study described in this report, we found that replication of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in the cell line Sf9, derived from the lepidopteran insect Spodoptera frugiperda, stimulated a DNA damage response, as indicated by an increased abundance of the S. frugiperda P53 protein (SfP53) and phosphorylation of the histone variant protein H2AX. Stimulation of the DNA damage response was dependent on viral DNA replication. Inhibition of the DNA damage response prevented both the increase in SfP53 accumulation and H2AX phosphorylation and also caused a 10- to 100-fold reduction in virus production, along with decreased viral DNA replication and late gene expression. However, silencing of Sfp53 expression by RNA interference did not significantly affect AcMNPV replication or induction of apoptosis by a mutant of AcMNPV lacking the antiapoptotic gene p35, indicating that these processes are not dependent on SfP53 in Sf9 cells.

Autographa californica multiple nucleopolyhedrovirus Ac92 (ORF92, P33) is required for budded virus production and multiply enveloped occlusion-derived virus formation.

The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNA-transfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C(155)XXC(158) amino acids, important for sulfhydryl oxidase activity, were mutated to A(155)XXA(158). The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype.

Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells.

Baculovirus-infected larvae release progeny viral occlusion bodies (OBs) to enable cyclical virus transmission to new hosts. The alphabaculovirus chitinase and cathepsin enzymes cause terminal liquefaction of host insect cadavers, aiding OB dispersal. The mechanism of cell lysis required to release the OBs is unclear but here we show Autographa californica multiple nucleopolyhedrovirus cathepsin protease activity is required for efficient release of the host tissue-degrading chitinase and cathepsin enzymes and critical for release of progeny OBs from virus-infected cells. Comparisons between viruses containing or lacking cathepsin indicate that cathepsin was necessary for OB release into cultured cell media or hemolymph of insects. In addition, pharmacological inhibition of cysteine protease activity in cells during infection blocked maturation of active cathepsin and OB release from infected cells. Together, these results suggest an important link between baculovirus-induced cell lysis, the concomitant maturation of cathepsin, and cellular release of chitinase, cathepsin and progeny OBs from cells.

Mutation of juxtamembrane cysteines in the tetraspanin CD81 affects palmitoylation and alters interaction with other proteins at the cell surface.

Palmitoylation of tetraspanins affects protein-protein interactions, suggesting a key role in the assembly of the tetraspanin web. Since palmitoylation occurs on intracellular cysteine residues, we examined whether mutating these residues in the human tetraspanin CD81 would affect the association of CD81 with other surface membrane proteins. Mutation of at least six of the eight juxtamembrane cysteines was required to completely eliminate detectable CD81 palmitoylation, indicating that several sites can be palmitoylated. Interestingly, these mutated proteins exhibited reduced cell surface detection by antibody compared to wild-type CD81, but this was not due to differences in the level of protein expression, trafficking to the cell surface, protein stability, or anti-CD81 antibody binding affinity. Instead, the mutant CD81 proteins appeared to be partially hidden from detection by anti-CD81 antibody, presumably due to altered interactions with other proteins at the cell surface. Associations with the known CD81-interacting proteins CD9 and EWI-2 were also impaired with the mutant CD81 proteins. Taken together, these findings indicate that mutation of juxtamembrane cysteines alters the interaction of CD81 with other proteins, either because of reduced palmitoylation, structural alterations in the mutant proteins, or a combination of both factors, and this affects the CD81 microenvironment on the cell surface.

Effects of Manipulating Fibroblast Growth Factor Expression on Sindbis Virus Replication In Vitro and in Aedes aegypti Mosquitoes.

Fibroblast growth factors (FGFs) are conserved among vertebrate and invertebrate animals and function in cell proliferation, cell differentiation, tissue repair, and embryonic development. A viral fibroblast growth factor (vFGF) homolog encoded by baculoviruses, a group of insect viruses, is involved in escape of baculoviruses from the insect midgut by stimulating basal lamina remodeling. This led us to investigate whether cellular FGF is involved in the escape of an arbovirus from mosquito midgut. In this study, the effects of manipulating FGF expression on Sindbis virus (SINV) replication and escape from the midgut of the mosquito vector Aedes aegypti were examined. RNAi-mediated silencing of either Ae. aegypti FGF (AeFGF) or FGF receptor (AeFGFR) expression reduced SINV replication following oral infection of Ae. aegypti mosquitoes. However, overexpression of baculovirus vFGF using recombinant SINV constructs had no effect on replication of these viruses in cultured mosquito or vertebrate cells, or in orally infected Ae. aegypti mosquitoes. We conclude that reducing FGF signaling decreases the ability of SINV to replicate in mosquitoes, but that overexpression of vFGF has no effect, possibly because endogenous FGF levels are already sufficient for optimal virus replication. These results support the hypothesis that FGF signaling, possibly by inducing remodeling of midgut basal lamina, is involved in arbovirus midgut escape following virus acquisition from a blood meal.

Inhibition of dicer activity in lepidopteran and dipteran cells by baculovirus-mediated expression of Flock House virus B2.

Prior studies have suggested that insect DNA viruses are negatively affected by dicer-2-mediated RNA interference (RNAi). To examine this further, we utilized an in vitro assay to measure dicer activity in lepidopteran and dipteran cells, combined with baculoviruses expressing the RNAi suppressor B2 from Flock House virus or Aedes aegypti dicer-2 (Aedicer-2) using a constitutive heat shock promoter. Addition of cell lysates containing baculovirus-expressed B2 to lysates from dipteran (S2, Aag2) or lepidopteran (Sf9) cells inhibited endogenous dicer activity in a dose-dependent manner, while expression of Aedicer-2 restored siRNA production in Ae. albopictus C6/36 cells, which are dicer-2 defective. However, B2 expression from the constitutive heat shock promoter had no impact on baculovirus replication or virulence in cell lines or larvae that were either highly permissive (Trichoplusia ni) or less susceptible (Spodoptera frugiperda) to infection. We determined that this constitutive level of B2 expression had little to no ability to suppress dicer activity in cell lysates, but higher expression of B2, following heat shock treatment, inhibited dicer activity in all cells tested. Thus, we cannot rule out the possibility that optimized expression of B2 or other RNAi suppressors may increase baculovirus replication and expression of heterologous proteins by baculoviruses.

Defective levels of both circulating dendritic cells and T-regulatory cells correlate with risk of recurrence in cutaneous melanoma.

BACKGROUND: Immune markers in the peripheral blood of melanoma patients provide useful information for clinical management although there is poor consensus on circulating cells which could putatively reflect the disease activity and play a prognostic role. Here, we investigated both dendritic cells (DCs) and T-regulatory cells (Tregs). METHODS: The number of DC subsets as myeloid (m) and plasmacytoid was measured by flowcytometry in 113 melanoma patients in different clinical stages and correlated with the disease activity to evaluate the recurrence free survival (RFS) calculated as difference between baseline and post-surgical values in relation to the criteria for the melanoma staging, as primary tumor removal, sentinel lymph node biopsy and completion of lymph node dissection. RESULTS: Circulating mDC levels were significantly lower in metastatic melanoma than in other stages and inversely correlated to Treg values while both populations were similarly expressed in inactive disease at stage I-III. Furthermore, the levels of these cells after melanoma removal were apparently related to the disease activity since their persistent defect reflected high risk of recurrence and reduced the RFS. CONCLUSIONS: This work highlighted the role of immune cell measurement for the management of melanoma activity and the identification of patients at potential risk of recurrence based on the mDC ratio.

Multiple human pressures in coastal habitats: variation of meiofaunal assemblages associated with sewage discharge in a post-industrial area.

Marine ecosystems are globally threatened by human activities, but some areas, such as those affected by abandoned industrial plants, show an overlap of acute and chronic impacts, which determine a considerable deterioration of their health status. Here we report the results of a research conducted on coastal sewers that discharge their loads in the highly contaminated area of Bagnoli-Coroglio (Tyrrhenian Sea, Western Mediterranean). The sampling area is characterized by heavy industrial activities (a steel plant using coal, iron and limestone) started in 1905 and ceased in 1990, which left widespread heavy metals and hydrocarbon contamination. After taking into account the potential influence of sediment grain size ranges through their inclusion as covariates in the analysis, we tested the potential impact of sewage discharge on the total abundance and multivariate structure of meiofaunal assemblages, as well as on the abundance of single taxa. The organic matter was analysed in terms of total phytopigment and biopolymeric carbon concentrations. Nematoda, Copepoda (including their nauplii), and Tardigrada were the most abundant meiofaunal taxa at all sites, but nematodes did not show a consistent pattern relative to the sewage outfalls. However, the sewer located in the historically most contaminated area showed a minimal abundance of all taxa, including nematodes, while copepods were relatively less abundant at the two southernmost sewers. Comparing the north vs. south site of the sewers, higher meiofaunal abundances were observed in the southward part, likely as a result of the local circulation. The results of this study indicate the general adaptation of meiofauna to multiple stressors (sewage discharge, superimposed to chronic industrial contamination) and its likely modulation by other local processes. They also provide relevant baseline information for future restoration interventions that would take into account the spatial variation of target organisms as needed.

Effects of temperature and shear force on infectivity of the baculovirus Autographa californica M nucleopolyhedrovirus.

Virus stability and infectivity during stressful conditions was assessed to establish guidelines for future virus filtration experiments and to contribute to the body of knowledge on a widely used virus. A recombinant baculovirus of Autographa californica M nucleopolyhedrovirus (AcMNPV), vHSGFP, was incubated at 15-65 degrees C. A 2-log decrease in virus infectivity occurred after virus incubation above 45 degrees C. The activation energy of virus deactivation was circa 108 kJ/mol. Dynamic light scattering revealed an increase in apparent virus particle size from 150+/-19 to 249+/-13 nm at 55 degrees C. Protein and DNA concentrations in solution correlated well with virus aggregation as temperature was increased. Infectivity of vHSGFP stored for 5 months at 4 degrees C or exposed to shear stress from stirring (100 rpm, 1.02x10(-5) psi) and pumping (50-250 ml/min, 1.45x10(-5) to 7.25x10(-5) psi) did not change with time. Unlike temperature variations, cold storage and shear stress appeared to have little impact on infectivity.

Analysis and functional annotation of expressed sequence tags from the fall armyworm Spodoptera frugiperda.

BACKGROUND: Little is known about the genome sequences of lepidopteran insects, although this group of insects has been studied extensively in the fields of endocrinology, development, immunity, and pathogen-host interactions. In addition, cell lines derived from Spodoptera frugiperda and other lepidopteran insects are routinely used for baculovirus foreign gene expression. This study reports the results of an expressed sequence tag (EST) sequencing project in cells from the lepidopteran insect S. frugiperda, the fall armyworm. RESULTS: We have constructed an EST database using two cDNA libraries from the S. frugiperda-derived cell line, SF-21. The database consists of 2,367 ESTs which were assembled into 244 contigs and 951 singlets for a total of 1,195 unique sequences. CONCLUSION: S. frugiperda is an agriculturally important pest insect and genomic information will be instrumental for establishing initial transcriptional profiling and gene function studies, and for obtaining information about genes manipulated during infections by insect pathogens such as baculoviruses.

Infection pattern and transmission potential of chikungunya virus in two New World laboratory-adapted Aedes aegypti strains.

Chikungunya virus (CHIKV) is an emerging mosquito-borne virus belonging to the Togaviridae, which is transmitted to humans by Aedes aegypti and Ae. albopictus. We describe the infection pattern of CHIKV in two New World Ae. aegypti strains, HWE and ORL. Both mosquito strains were susceptible to the virus but showed different infection patterns in midguts and salivary glands. Even though acquisition of a bloodmeal showed moderate levels of apoptosis in midgut tissue, there was no obvious additional CHIKV-induced apoptosis detectable during midgut infection. Analysis of expression of apoptosis-related genes suggested that CHIKV infection dampens rather than promotes apoptosis in the mosquito midgut. In both mosquito strains, the virus was present in saliva within two days post-oral infection. HWE and ORL mosquitoes exhibited no salivary gland infection barrier; however, only 60% (HWE) to 65% (ORL) of the females had released the virus in their saliva at one week post-oral acquisition, suggesting a salivary gland escape barrier. CHIKV induced an apoptotic response in salivary glands of HWE and ORL mosquitoes, demonstrating that the virus caused pathology in its natural vector.

Expression of the Cydia pomonella granulovirus matrix metalloprotease enhances Autographa californica multiple nucleopolyhedrovirus virulence and can partially substitute for viral cathepsin.

The Cydia pomonella granulovirus open reading frame 46 (CpGV-ORF46) contains predicted domains found in matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. We showed that CpGV-MMP was active in vitro. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing CpGV-ORF46 replicated similarly to a control virus lacking CpGV-ORF46 in cultured cells. The effects of AcMNPV expressing CpGV-MMP on virus infection in cultured cells and Trichoplusia ni larvae in the presence or absence of other viral degradative enzymes, cathepsin and chitinase, were evaluated. In the absence of cathepsin and chitinase or cathepsin alone, larval time of death was significantly delayed. This delay was compensated by the expression of CpGV-MMP. CpGV-MMP was also able to promote larvae melanization in the absence of cathepsin and chitinase. In addition, CpGV-MMP partially substituted for cathepsin in larvae liquefaction when chitinase, which is usually retained in the endoplasmic reticulum, was engineered to be secreted.